Receptor ligands and methods of modulating receptors

ABSTRACT

The invention provides natural ligands of various receptors and methods of identifying modulators of various receptors using the ligands. Methods of using the modulators to treat diseases or disorder associated with dysfunction of the receptor are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application60/424,093, filed Nov. 5, 2002, which application is herein incorporatedby reference.

FIELD OF THE INVENTION

The invention relates to the identification of specific ligands thatbind previously identified G-protein coupled receptors (GPCRs) andmethods for identifying and using modulators of the receptor-ligandinteractions for various therapeutic indications.

BACKGROUND OF THE INVENTION

G-protein coupled receptors are cell surface receptors that indirectlytransduce extracellular signals to downstream effectors, e.g.,intracellular signaling proteins, enzymes, or channels. Changes in theactivity of these effectors then mediate subsequent cellular events. Theinteraction between the receptor and the downstream effector is mediatedby a G-protein, a heterotrimeric protein that binds GTP. Examples ofmammalian G proteins include Gi, Go, Gq, Gs, and Gt.

G-protein coupled receptors (“GPCRs”) typically have seven transmembraneregions, along with an extracellular domain and a cytoplasmic tail atthe C-terminus. These receptors form a large superfamily of relatedreceptor molecules that play a key role in many signaling processes,such as sensory and hormonal signal transduction. The furtheridentification of GPCRs and the natural ligands of the receptors isimportant for understanding the normal process of signal transductionand as well as its involvement in pathologic processes. For example,GPCRs can be used for disease diagnosis as well as for drug discovery.GPCR ligands may be used for the treatment of GPCR-related disorders andfor the identification of additional modulators of GPCR activity.Further identification of GPCRs and ligands that bind to GPCRs istherefore of great interest.

SUMMARY OF THE INVENTION

The present invention relates to the identification and characterizationof ligands for particular G protein-coupled receptors (GPCRs) andmethods for identifying and using modulators of the receptor-ligandinteractions. Specifically, the inventors have shown that a naturalligand for TGR2 is β-alanine, a natural ligand for GPR77 is acylationstimulating protein (ASP), and a natural ligand for LGR4, LGR5, or LGR6is a stanniocalcin. Furthermore, the inventors have discovered thatshort chain fatty acids, typically of 2-3 carbons in length, activateGPR43 and medium and long chain fatty acids that are 6 carbons orgreater in length activate GPR40. Lastly, the invention is also based onthe discovery that succinic acid is a natural ligand for TGR18 and thatα-ketoglutaric acid is a natural ligand for TGR164. Modulators of thereceptor-ligand interaction may be used, for example, for the treatmentof a disease or condition associated with a TGR2, GPR77, LGR4, LGR5,LGR6, GPR40, GPR43, TGR18, or TGR164.

Thus, the current invention provides a method of identifying a modulatorof a TGR2 polypeptide that has G-protein coupled receptor activity and(A) comprises at least 70% amino acid sequence identity to SEQ ID NO:2,(B) comprises at least 50 contiguous amino acids of SEQ ID NO:2, or (C)comprises the amino acid sequence of SEQ ID NO:2; wherein the methodcomprises: contacting a compound with the TGR2 polypeptide; anddetermining the level of binding of β-alanine to the TGR2 polypeptide incomparison to the level of binding in the absence of the compound. Insome embodiments, the TGR2 polypeptide consists of at least 50, often atleast 40, 30, or 20, contiguous amino acids of SEQ ID NO:2. Often, theTGR2 is recombinant. The step of determining the level of binding cancomprise a binding assay such as a competitive binding assay ordetecting an alteration in a β-alanine-induced TGR2 activity such asinositol phosphate accumulation.

Modulators of TGR2 receptor-ligand interactions can be used, forexample, in a method of treating a patient with a TGR2-associateddisorder, the method comprising administering a therapeuticallyeffective amount of a compound identified as set forth above. TheTGR2-associated disorder may be, but is not limited to, a pain disorder,a disorder in the immune system, or an inflammatory disorder.

In another aspect, the invention provides a method of identifying amodulator of a GPR77 polypeptide that has G-protein coupled receptoractivity and (A) comprises at least 70% amino acid sequence identity toSEQ ID NO:4, (B) comprises at least 50 contiguous amino acids of SEQ IDNO:4, or (C) comprises the amino acid sequence of SEQ ID NO:4; whereinthe method comprises: contacting a compound with the GPR77 polypeptide;and determining the level of binding of acylation stimulating protein(ASP) to the GPR77 polypeptide in comparison to the level of binding inthe absence of the compound. In some embodiments, the GPR77 polypeptideconsists of at least 50, often at least 40, 30, or 20, contiguous aminoacids of SEQ ID NO:4. Often, the GPR77 is recombinant.

The step of determining the level of binding may comprise detecting theability of the compound to modulate ASP binding in a competitive bindingassay. The step of determining the level of binding may also comprisedetecting an alteration in an ASP-induced GPR77 activity. In someembodiments, the method may further comprise a step of detecting analteration in ASP-induced triglyceride synthesis.

The invention also provides a method of treating a patient with aGPR77-associated disorder, the method comprising administering atherapeutically effective amount of a compound identified using themethod set forth above. The GPR77-associated disorder may be, but is notlimited to diabetes, obesity, or atherosclerosis.

In another aspect, the invention provides a method of identifying amodulator of an LGR4, LGR5, or LGR6 polypeptide that has G-proteincoupled receptor activity and (A) comprises at least 70% amino acidsequence identity to SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, (B)comprises at least 50 contiguous amino acids of SEQ ID NO:6, SEQ IDNO:8, or SEQ ID NO:10, or (C) comprises the amino acid sequence of SEQID NO:6, SEQ ID NO:8, or SEQ ID NO:10; wherein the method comprises:contacting a compound with the polypeptide; and determining the level ofbinding of a stanniocalcin to the polypeptide in comparison to the levelof binding in the absence of the compound. In some embodiments, the LGRpolypeptide consists of at least 50, often at least 40, 30, or 20,contiguous amino acids of SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.Often, the LGR4, LGR5, or LGR6 is recombinant. The step of determiningthe level of binding may comprise detecting the ability of the compoundto alter stanniocalcin binding in a competitive binding assay.Alternatively, the step of determining the level of binding may comprisedetecting an alteration in a stanniocalcin-induced activity of LGR4,LGR5, or LGR6. In some embodiments, the method may further comprisedetecting an alteration in stanniocalcin-induced changes in calcium orphosphate levels in a cell.

In another embodiment, the invention provides a method of treating apatient with an LGR4, LGR5, or LGR6-associated disorder, the methodcomprising administering a therapeutically effective amount of acompound identified as set forth above. The disorder may be, but is notlimited to, a disorder of calcium or phosphate metabolism; a bonedisorder; a kidney disorder; or a growth or reproductive disorder.

In another aspect, the invention provides a method of identifying amodulator of a GPR40 polypeptide that has G-protein coupled receptoractivity and (A) comprises at least 70% amino acid sequence identity toSEQ ID NO:12, (B) comprises at least 50 contiguous amino acids of SEQ IDNO:12, or (C) comprises the amino acid sequence of SEQ ID NO:12; whereinthe method comprises: contacting a compound and a medium or long chainfatty acid with the polypeptide; and determining the level of activityof the polypeptide, in comparison to the level of activity in theabsence of the compound. In some embodiments, the GPR40 polypeptideconsists of at least 50, often at least 40, 30, or 20, contiguous aminoacids of SEQ ID NO:12. Often, the fatty acid is a long chainpolyunsaturated fatty acid. Often, the GPR40 is recombinant. In someembodiments, the step of determining the level of activity comprisesdetermining the level of binding of the modulator, or the naturalligand, to the GPR40. In other embodiments, the method may comprisedetecting an alteration in fatty acid-induced Gq activity.

In another aspect, the invention provides a method of identifying amodulator of a GPR43 polypeptide that has G-protein coupled receptoractivity and (A) comprises at least 70% amino acid sequence identity toSEQ ID NO:14, (B) comprises at least 50 contiguous amino acids of SEQ IDNO:14, or (C) comprises the amino acid sequence of SEQ ID NO:14; whereinthe method comprises: contacting a compound with the polypeptide; anddetermining the level of activation of the polypeptide by a short chainfatty acid, preferably of 2-3 carbons in length, in comparison to thelevel of activation in the absence of the compound. In some embodiments,the GPR43 polypeptide consists of at least 50, often at least 40, 30 or20, contiguous amino acids of SEQ ID NO:14. Often, the fatty acid is along chain polyunsaturated fatty acid and the GPR43 is recombinant. Insome embodiments, the step of determining the level of activity maycomprise determining the level of binding of the modulator, or thenatural ligand, to the GPR43. In other embodiments, the method maycomprise detecting an alteration in fatty acid-induced Gq activity.

In another embodiment, the invention provides a method of treating apatient with a GPR40 or GPR43-associated disorder, the method comprisingadministering a therapeutically effective amount of a compoundidentified as set forth above. The disorder may be, but is not limitedto, a disorder relating to fat metabolism, such as obesity, diabetes,atherosclerosis, coronary artery disease, and stroke.

In another aspect, the invention provides a method of identifying amodulator of a TGR18 polypeptide that has G-protein coupled receptoractivity and (A) comprises at least 70% amino acid sequence identity toSEQ ID NO:16, (B) comprises at least 50 contiguous amino acids of SEQ IDNO:16, or (C) comprises the amino acid sequence of SEQ ID NO:16; whereinthe method comprises: contacting a compound with the polypeptide; anddetermining the level of activation of the polypeptide by succinic acidin comparison to the level of activation in the absence of the compound.In some embodiments, the TGR18 polypeptide consists of at least 50,often at least 40, 30 or 20, contiguous amino acids of SEQ ID NO:16.Often, the TGR18 is recombinant, e.g., encoded by SEQ ID NO:15. In someembodiments, the step of determining the level of activity may comprisedetermining the level of binding of the modulator, or the naturalligand, to the TGR18. In other embodiments, the method may comprisedetecting an alteration in succinic acid-induced GPCR activity, e.g., achange in GPCR-mediated gene activation, mobilization of intracellularcalcium, or an increase in inositol phosphate.

In another aspect, the invention provides a method of identifying amodulator of a TGR164 polypeptide that has G-protein coupled receptoractivity and (A) comprises at least 70% amino acid sequence identity toSEQ ID NO:18, (B) comprises at least 50 contiguous amino acids of SEQ IDNO:18, or (C) comprises the amino acid sequence of SEQ ID NO:18; whereinthe method comprises: contacting a compound with the polypeptide; anddetermining the level of activation of the polypeptide by α-ketoglutaricacid in comparison to the level of activation in the absence of thecompound. In some embodiments, the TGR164 polypeptide consists of atleast 50, often at least 40, 30 or 20, contiguous amino acids of SEQ IDNO:18. Often, the TGR164 is recombinant, e.g., encoded by SEQ ID NO:17.In some embodiments, the step of determining the level of activity maycomprise determining the level of binding of the modulator, or thenatural ligand, to the TGR164. In other embodiments, the method maycomprise detecting an alteration in α-ketoglutaric acid-induced GPCRactivity, e.g., a change in GPCR-mediated gene activation, mobilizationof intracellular calcium, or an increase in inositol phosphate.

In another embodiment, the invention provides a method of treating apatient with a TGR18 or TGR164-associated disorder, the methodcomprising administering a therapeutically effective amount of acompound identified as set forth above. The disorder may be, but is notlimited to, a kidney-associated disorder, e.g., renal failure,nephritis, glomerulonephritis, hypertension, and other diseases in whichthe kidney is dysfunctional.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a Aequorin assay using CHO cells transientlytransfected with TGR2.

FIG. 2 provides exemplary data showing that short chain fatty acidsactivate GPR43.

FIG. 3 provides exemplary data showing that medium and long chainsaturated fatty acids activate GPR40.

FIG. 4 provides exemplary data showing that polyunsaturated fatty acidsactivate GPR40.

FIG. 5 provides exemplary data showing that hypolipidemic drugs andfibrates activate GPR40.

FIGS. 6A, 6B, and 6C provide exemplary data showing that succinic acidand related compounds activate TGR18.

FIGS. 7A and 7B provide exemplary data showing that α-ketoglutaric acidand related compounds activate TGR164.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTSIntroduction

The current invention is based on the discovery of natural ligands forvarious GPCRs. The inventors have determined that β-alanine is a naturalligand for TGR2; acylation stimulating protein (ASP) is a natural ligandto GPR77; stanniocalcins are natural ligands for LGR4, LGR5, and LGR6;fatty acids that are 6 carbons or greater in length are natural ligandsfor GPR40, fatty acids of 2-3 carbons in length are natural ligands forGPR43; succinic acid is a natural ligand for TGR18; and α-ketoglutaricacid is a ligand for TGR164. Accordingly, these ligands, or analogs,conservative modifications, or variants thereof, may be used to modulateGPCR activity and for the treatment of diseases or conditions related toGPCR activity. Further, the ligands may be used to identify compoundsthat modulate ligand binding and activation of the cognate GPCR. Suchmodulators may be used to treat, for example, TGR2, GPR77, LGR4, LGR5,LGR6, GPR40, GPR43, TGR18, and TGR164-related disorders.

β-alanine is a naturally occurring amino acid that is a precursor ofco-enzyme A. It is typically present in synaptic particles and binds toreceptors for inhibitor neurotransmitters, e.g., glycine and GABA.Further, it may be released after NMDA receptor stimulation or undercell damaging conditions. Thus, it plays an important role in thenervous system and in the body's response to injury.

ASP is a potent lipogenic factor in mammals that simulatestriacylglycerol synthesis. It is identical to the desarginated form ofcomplement C3a, i.e., C3adesArg, but does not bind the C3a receptor(see, e.g., Murray et al., Biochem J. 342:41-48, 1999; Baldo, et al., J.Clin. Invest. 92:1543-1547, 1993). It is believed to play an importantrole in fat metabolism and diseases such as obesity, diabetes,atherosclerosis and other disorders in lipid metabolism.

Stanniocalcins are involved in mineral metabolism, e.g., calcium andphosphate regulation in mammals as well as other animals, e.g., fish.Two human sequences are known (see, e.g., Chang et al., Mol. Cell.Endocrinol. 112:241-247, 1995; WO 95/24411; U.S. Pat. No. 6,008,322; andU.S. Application Publication No. 20020042372). Typically, stanniocalcinscan regulate calcium and phosphate transport across mammalian intestinalepithelia. Stanniocalcins are believed to play a role in bone disordersor other disorders related to calcium and phosphate metabolism, as wellas growth and reproduction.

The GPCRs GPR41 and GPR42 bind to endogenous fatty acid ligands of 3-5carbons in length (see, WO 01/61359). The current inventors havediscovered that medium and long chain fatty acids, in particular fattyacids having a carbon chain length of six or greater are natural ligandsfor GPR40, and further, that fatty acids of 2-3 carbons are naturalligands for GPR43.

TGR18 has 38% amino acid identity to P2Y receptors; however, P2Yligands, e.g., extracellular adenosine nucleotides, do not activateTGR18. Similarly, TGR164 shares 50% amino acid identity with TGR18 andalso belongs to the P2Y family of GPCRs. The current inventors havediscovered that succinic acid and analogs, e.g., succinic anhydride,maleic acid, oxalacetic acid, methylmalonic acid and itaconic acid areligands for TGR18; and that α-ketoglutaric acid and analogs, e.g.,itaconic acid, are ligands for TGR164.

The present invention thus provides nucleic acids encoding G proteincoupled receptors TGR2, GPR77, LGR4, LGR5, LGR6, GPR40, GPR43, TGR18,and TGR164; and natural ligands of these GPCRs. The GPCR nucleic acidand protein sequences provide means for assaying for and identifyingmodulators of ligand binding and ligand-mediated GPCR signaltransduction, e.g., activators, inhibitors, stimulators, enhancers,agonists, and antagonists. Such modulators are useful forpharmacological modulation of signaling pathways, e.g., in cells andtissues that express TGR2, GPR77, LGR4, LGR5, LGR6, GPR40, GPR41, TGR18,or TGR164.

Exemplary TGR2 nucleic acid and protein sequences are provided hereinand have been described (see, e.g., WO 01/66750; WO 01/36471; WO01/83748; WO 01/70814; WO 01/57085; WO 01/48188; EP1178053; WO 01/98330;and WO 01/83555).

Exemplary GPR77 nucleic acid and protein sequences are provided hereinand have been described (see, e.g., WO 01/49847; WO 01/62797; WO01/57085; WO 01/36471; EP1094076; WO 01/48189; WO 01/55338; WO 01/57190;and WO 00/14229).

Exemplary GPR40 nucleic acid and protein sequences are provided hereinand have been described (see, e.g., WO 00/22129; and WO 02057783).

Exemplary GPR43 nucleic acid and protein sequences are provided hereinand have been described (see, e.g., U.S. Pat. No. 5,910,430; WO00/22129; WO 99/15656; and WO 98/40483).

Exemplary TGR18 nucleic acid and protein sequences are provided hereinand have been described (see, e.g., U.S. Pat. Nos. 5,871,963 and6,063,582; WO 00/31258; WO 97/24929; WO 00/22131; WO 01/98351; WO00/61628; and WO 01/79449).

Exemplary TGR164 nucleic acid and protein sequences are provided hereinand have been described (see, e.g., WO 01/36471; WO 01/87937; WO01/49847; WO 01/87980; WO 02/14511; GB2365012; WO 02/46414; EP1219638;WO 01/36473; and WO 0157190).

The GPCRs described herein exhibit patterns of tissue-specificexpression. Such tissue specific expression indicates that modulatorsidentified using the methods of the invention can be used tospecifically modulate GPCR activity in particular cell types. Forexample, human TGR2 is expressed in the immune system and in neuronaltissue, e.g., pain sensory neurons. Thus, TGR2 modulators, e.g.,β-alanine or other modulators that modulate the level of β-alaninebinding to the receptor or β-alanine-induced TGR2 activity, may be usedfor the treatment of immune or inflammatory disorders, or for thetreatment of pain, e.g., chronic or acute pain, including chronic painsyndromes. Pain disorders include, but are not limited, to neuropathicpain resulting from injury to specific nerves; pain associated withcancer, such as pain resulting from bone metastases in cancer, painassociated with chronic or acute inflammatory diseases, and painassociated with chronic ganglionic viral infections, e.g., Herpesinfection.

Similarly, GPR77 is expressed in the brain, e.g., the hypothalamus andcortex, adipose and other tissues that are involved in fat metabolism,e.g., liver. Modulators of ASP binding to GPR77 or ASP-induced GPR77activity can be used, for example, for the treatment of diseases orconditions that involve hypothalamus dysfunction or disorders of fatmetabolism, e.g., obesity. LGR4, LGR5, and LGR6 are expressed in varioustissues, including reproductive organs. Modulators of stanniocalcinbinding or stanniocalcin-induced LGR4, LGR5, or LGR6 activity can beused, for example, for the treatment of diseases or conditions thatinvolve mineral metabolism, e.g., calcium and phosphate metabolism, suchas osteoporosis, as well as growth and reproductive disorders.

TGR18 and TGR164 are expressed predominantly in the kidney. Modulatorsof TGR18 and TGR164 may therefore be used, for example, for thetreatment of kidney diseases or diseases related to kidney function.Kidney diseases or diseases related to kidney function include, but arenot limited to glomerulonephritis, scarring glomerular disease, renaldiseases having an inflammatory component, renal diseases having aglomerular extracellular matrix accumulation component, proteinuria,microaneurysm formation, hypertension, and other diseases that involverenal failure.

Modulators that compete with the binding and/or activity of theparticular fatty acid endogenous ligands for GPR 40 and GPR 43 can beused to treat various diseases associated with disorders of fatmetabolism including, but not limited to, coronary artery disease,atherosclerosis, thrombosis, obesity, diabetes, stroke, and othervascular diseases.

The GPCR ligands identified herein and modulators of ligand binding andGPCR activity can also be used to further study signal transduction.Thus, the invention provides assays for signal transduction modulation,where the GPCRs act as direct or indirect reporter molecules for theeffect of modulators on ligand-mediated signal transduction. GPCRs canbe used in assays in vitro, ex vivo, and in vivo, e.g., to measurechanges in transcriptional activation of GPCRs; ligand binding;phosphorylation and dephosphorylation; GPCR binding to G-proteins;G-protein activation; regulatory molecule binding; voltage, membranepotential, and conductance changes; ion flux; changes in intracellularsecond messengers such as cAMP, diacylglycerol, and inositoltriphosphate; and changes in intracellular calcium levels.

Methods of assaying for modulators of ligand binding and signaltransduction include in vitro ligand binding assays using the GPCRs,portions thereof such as the extracellular domain, or chimeric proteinscomprising one or more domains of a GPCR, oocyte GPCR expression ortissue culture cell GPCR expression, either naturally occurring orrecombinant; membrane expression of a GPCR, either naturally occurringor recombinant; tissue expression of a GPCR; expression of a GPCR in atransgenic animal, etc.

Related GPCR genes, e.g., from other species should share at least about70%, 80%, 90%, or greater, amino acid identity over a amino acid regionat least about 25 amino acids in length, optionally 50 to 100 aminoacids in length.

Specific regions of the GPCR nucleotide and amino acid sequences may beused to identify polymorphic variants, interspecies homologs, andalleles of GPCRs. This identification can be made in vitro, e.g., understringent hybridization conditions or PCR (using primers that hybridizeto SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, or 17), and sequencing, or byusing the sequence information in a computer system for comparison withother nucleotide sequences. Typically, identification of polymorphicvariants and alleles of a GPCR is made by comparing an amino acidsequence of about 25 amino acids or more, e.g., 50-100 amino acids.Amino acid identity of approximately at least 70% or above, optionally75%, 80%, 85% or 90-95% or above typically demonstrates that a proteinis a polymorphic variant, interspecies homolog, or allele of a GPCR.Sequence comparison is performed using the BLAST and BLAST 2.0 sequencecomparison algorithms with default parameters, discussed below.Antibodies that bind specifically to a GPCR or a conserved regionthereof can also be used to identify alleles, interspecies homologs, andpolymorphic variants. The polymorphic variants, alleles and interspecieshomologs are expected to retain the seven transmembrane structure of aG-protein coupled receptor.

Definitions

“GPCR,” “TGR”, “TGR2”, “GPR77”, “LGR4”, “LGR5”, “LGR6”, “GPR40”,“GPR43”, “TGR18”, or “TGR164” all refer to G-protein coupled receptors,the genes for most of which have been mapped to particular chromosomesand which are expressed in particular cell types. These GPCRs have seventransmembrane regions and have “G-protein coupled receptor activity,”e.g., they bind to G-proteins in response to extracellular stimuli andpromote production of second messengers such as diacylglycerol (DAG),IP₃, cAMP, and Ca²⁺ via stimulation of downstream effectors such asphospholipase C and adenylate cyclase (for a description of thestructure and function of GPCRs, see, e.g., Fong, supra, and Baldwin,supra).

Topologically, GPCRs have an N-terminal “extracellular domain,” a“transmembrane domain” comprising seven transmembrane regions andcorresponding cytoplasmic and extracellular loops, and a C-terminal“cytoplasmic domain” (see, e.g., Buck & Axel, Cell 65:175-187 (1991)).These domains can be structurally identified using methods known tothose of skill in the art, such as sequence analysis programs thatidentify hydrophobic and hydrophilic domains (see, e.g., Kyte &Doolittle, J. Mol. Biol. 157:105-132 (1982)). Such domains are usefulfor making chimeric proteins and for in vitro assays of the invention.

The terms “GPCR” and “TGR2”, “GPR77”, “LGR4”, “LGR5”, “LGR6”, “GPR40”,“GPR43”, “TGR18”, or “TGR164” therefore refer to nucleic acid andpolypeptide polymorphic variants, alleles, mutants, and interspecieshomologs and GPCR domains thereof that: (1) have an amino acid sequencethat has greater than about 65% amino acid sequence identity, 70%, 75%,80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%or greater amino acid sequence identity, preferably over a window of atleast about 25, 50, 100, 200, 500, 1000, or more amino acids, to asequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16 or 18; (2) bind toantibodies raised against an immunogen comprising an amino acid sequenceof SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16 or 18 and conservativelymodified variants thereof; (3) have at least 15 contiguous amino acids,more often, at least 20, 30, 40, 50, 100, 200, or 300, contiguous aminoacids of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16 or 18; (4) specificallyhybridize (with a size of at least about 100, preferably at least about500 or 1000 nucleotides) under stringent hybridization conditions to asequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15 or 17 and conservativelymodified variants thereof; (5) have a nucleic acid sequence that hasgreater than about 95%, preferably greater than about 96%, 97%, 98%,99%, or higher nucleotide sequence identity, preferably over a region ofat least about 50, 100, 200, 500, 1000, or more nucleotides, to SEQ IDNO:1, 3, 5, 7, 9, 11, 13, 15, or 17; or (6) are amplified by primersthat specifically hybridize under stringent conditions to SEQ ID NO:1,3, 5, 7, 9, 11, 13, 15, or 17. This term also refers to a domain of aGPCR, as described above, or a fusion protein comprising a domain of aGPCR linked to a heterologous protein. A GPCR polynucleotide orpolypeptide sequence of the invention is typically from a mammalincluding, but not limited to, human, mouse, rat, hamster, cow, pig,horse, sheep, or any mammal. A “TGR2, GPR77, LGR4, LGR5, LGR6, GPR40,GPR43, TGR18, or TGR164 polynucleotide” and a “TGR2, GPR77, LGR4, LGR5,LGR6, GPR40, GPR43, TGR18, or TGR164 polypeptide,” are both eithernaturally occurring or recombinant.

A “full length” TGR2, GPR77, LGR4, LGR5, LGR6, GPR40, GPR43, TGR18, orTGR164” protein or nucleic acid refers to a polypeptide orpolynucleotide sequence, or a variant thereof, that contains all of theelements normally contained in one or more naturally occurring, wildtype TGR2, GPR77, LGR4, LGR5, LGR6, GPR40, GPR43, TGR18, or TGR164polynucleotide or polypeptide sequences. It will be recognized, however,that derivatives, homologs, and fragments of TGR2, GPR77, LGR4, LGR5,LGR6, GPR40, GPR43, TGR18, or TGR164 can be readily used in the presentinvention.

In some embodiments, the GPCR used in the methods of the invention is afragment or domain that essentially consists of, at least 15, often atleast 20, 30, 40, or 50, contiguous amino acids of SEQ ID NO:2, 4, 6, 8,10, 12, 14, 16, or 18.

“Extracellular domain” refers to the domain of a GPCR that protrudesfrom the cellular membrane and often binds to an extracellular ligand.This domain is often useful for in vitro ligand binding assays, bothsoluble and solid phase.

“Transmembrane domain,” comprises seven transmembrane regions plus thecorresponding cytoplasmic and extracellular loops. Certain regions ofthe transmembrane domain can also be involved in ligand binding.

“Cytoplasmic domain” refers to the domain of a GPCR that protrudes intothe cytoplasm after the seventh transmembrane region and continues tothe C-terminus of the polypeptide.

The term “β-alanine” refers to a naturally occurring amino acid that isa precursor of co-enzyme A synthesis. A “β-alanine” as used hereinincludes amino acid analogs and amino acid mimetics that bind to TGR2and function in a manner similar to the naturally occurring β-alanine.β-alanine is typically present in synaptic particles and binds toreceptors for inhibitor neurotransmitters, e.g., glycine and GABA. Themolecules are also present in plasma and cerebrospinal fluid (CSF) at μMconcentrations.

“Acylation stimulating protein” or “ASP” is identical to thedesarginated form of complement C3a, i.e., C3adesArg (see, e.g., Murrayet al., Biochem J. 342:41-48, 1999; Baldo, et al., J. Clin. Invest.92:1543-1547; 1993). “ASP” sequences are known (e.g., U.S. Pat. No.5,714,466). As used herein, “ASP” also refers to homologs, variants ormutants that bind to GPR77, but typically not to C3a, and preferably,that stimulate triacylglyceride synthesis as described, for example, inU.S. Pat. No. 5,714,466. Typically, an “ASP” binds to GPR77 and has anamino acid sequence that has greater than about 65% amino acid sequenceidentity, often 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity to anASP as set forth in U.S. Pat. No. 5,714,466; or binds to antibodiesraised against an immunogen comprising an ASP as set forth in U.S. Pat.No. 5,714,466.

As used herein, “stanniocalcin” refers to a polypeptide, and allelicvariants, homologs, mutants and fragments thereof, that binds to LGR4,LGR5, or LGR6; and has an amino acid sequence that has greater thanabout 65% amino acid sequence identity, often 70%, 75%, 80%, 85%, 90%,preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greateramino acid sequence identity to the human stanniocalcin sequences shownin FIG. 1 in U.S. Pat. No. 6,008,322; or binds to antibodies raisedagainst an immunogen comprising a human stanniocalcin amino acidsequence shown in FIG. 1 of U.S. Pat. No. 6,008,322.

A “medium or long chain fatty acid” as used herein refers to a fattyacid having a chain length of at least six carbons. Often, a “medium orlong chain fatty acid” used in the invention has a chain length greaterthan six carbons, for example eight, ten, twelve, fourteen, sixteen,eighteen, twenty, or twenty two carbons in length.

A “short chain fatty acid” generally refers to a fatty acid having achain length of five carbons or less. A “short chain fatty acid” ligandfor GPR43 is typically 2-3 carbons in length.

The term “fatty acid” as used herein encompasses both saturated andpolyunsaturated fatty acids and isomeric forms, and also includes short,medium, and long chain fatty acids. Examples of fatty acids are setforth in Tables I, II, and III, infra. TABLE ICH₃—(CH₂)_(f)—(CH═CH)_(g)(CH₂)_(h)CO₂H Carbons f g h Acid Name 16 5 1 7Palmitoleic 18 7 1 7 Oleic 18 10 1 4 Petroselenic 18 5 1 9 Vaccenic 18 33 7 Punicic 18 1 4 7 Parinaric 20 9 1 7 Gadoleic 22 9 1 9 Cetoleic

TABLE II CH₃—(CH₂)_(n)—(CH═CH—CH₂)_(m)—(CH₂)_(p)—CO₂H Carbons n m p AcidName 18 4 2 6 Linoleic 18 1 3 6 Linolenic 20 4 4 2 Arachidonic

TABLE III CH₃—(CH₂)_(w)—CO₂H Carbons w Acid Name 12 10 Lauric 14 12Myristic 16 14 Palmitic 18 16 Stearic 20 18 Eicosanoic 22 20 Docosanoic

It will be appreciated that the unsaturated fatty acids occur inisomeric forms due to the presence of the one or more unsaturatedpositions. The fatty acids of the present invention are intended toinclude both the individual double bond isomers as well as mixturesthereof. For example, although, docosatetraenoic acid anddocosapentaenoic acid have the same number of carbons, they aredifferent isomeric forms.

A “fibrate” is a member of a class of lipid lowering drugs that arefibric acid derivatives (e.g., gemfibrozil, clofibrate, fenofibrate).The fibric acid derivatives are lipid regulating drugs that promote thecatabolism of triglyceride-rich lipoproteins, secondary to theactivation of lipoprotein lipase, and promote the reduction of apoC-IIIsynthesis. Fibrates are well known in the art (see, e.g., U.S. Pat. No.4,058,552). Examples of the fibrate compounds include bezafibrate,beclobrate, binifibrate, ciplofibrate, clinofibrate, clofibrate,clofibric acid, etofibrate, fenofibrate, gemfibrozil, nicofibrate,pirifibrate, ronifibrate, simfibrate, theofibrate, etc.

A “TGR18 ligand” as used herein refers to a compound that activatesTGR18, such as any alpha,omega-dicarboxylic acid alkane, or precursorthereto or salt thereof, wherein the alkane is a methylene, ethylene orethenylene group, and wherein the methylene, ethylene and ethenylenegroups can be substituted or incorporated into an aromatic ornon-aromatic ring. A “TGR18 ligand” is oriented such that it binds toTGR18. Examples of TGR18 ligands include, but are not limited to,succinic acid, succinyl anhydride, maleic acid, oxalacetic acid,methylmalonic acid and itaconic acid. Binding of a TGR18 ligand istypically determined by measuring GPCR activity.

A “TGR164 ligand” as used herein refers to a compounds that activatesTGR164, such as any alpha,omega-dicarboxylic acid alkane, or precursorthereto or salt thereof, wherein the alkane is an ethylene or propylenegroup, and wherein the ethylene and propylene groups can be substitutedwith suitable substituents, and the ethylene group can be incorporatedinto a non-aromatic ring. In the TGR164 ligands described herein, thealpha position is substituted with a pi-system such that the pi-systemdoes not hydrogen bond to the carboxylic acid at the omega position, andsuch that the two carboxylic acids are oriented so as to bind to TGR164.Examples of compounds that activate TGR164 include, but are not limitedto, alpha-ketoglutaric acid and itaconic acid. Binding of a TGR164ligand is typically determined by measuring GPCR activity.

“GPCR activity” refers to the ability of a GPCR to transduce a signal.Such activity can be measured, e.g., in a heterologous cell, by couplinga GPCR (or a chimeric GPCR) to a G-protein and a downstream effectorsuch as PLC or adenylate cyclase, and measuring increases inintracellular calcium (see, e.g., Offermans & Simon, J. Biol. Chem.270:15175-15180 (1995)). Receptor activity can be effectively measuredby recording ligand-induced changes in [Ca²⁺]_(i) using fluorescentCa²⁺-indicator dyes and fluorometric imaging. A “natural ligand-inducedactivity” as used herein, refers to activation of the GPCR by a naturalligand of the GPCR. Activity can be assessed using any number ofendpoints to measure the GPCR activity. For example, activity of a GPCRas disclosed herein, such as a TGR2, GPR40, GPR43, TGR18, or TGR164, maybe assessed using an assay such as calcium mobilization, e.g., anAequorin assay, or inositol phosphate accumulation.

A “host cell” is a naturally occurring cell or a transformed cell thatcontains an expression vector and supports the replication or expressionof the expression vector. Host cells may be cultured cells, explants,cells in vivo, and the like. Host cells may be prokaryotic cells such asE. coli, or eukaryotic cells such as yeast, insect, amphibian, ormammalian cells such as CHO, HeLa, and the like.

“Biological sample” as used herein is a sample of biological tissue orfluid that contains GPCR nucleic acids or polypeptides. Such samplesinclude, but are not limited to, tissue isolated from humans, mice, andrats. Biological samples may also include sections of tissues such asfrozen sections taken for histologic purposes. A biological sample istypically obtained from a eukaryotic organism, such as insects,protozoa, birds, fish, reptiles, and preferably a mammal such as rat,mouse, cow, dog, guinea pig, or rabbit, and most preferably a primatesuch as chimpanzees or humans. Preferred tissues typically depend on theknown expression profile of the GPCR, and include e.g., normal colon,spleen, kidney, liver, hypothalamus, adipose, or other tissues.

The phrase “functional effects” in the context of assays for testingcompounds that modulate GPCR-mediated signal transduction includes thedetermination of any parameter that is indirectly or directly under theinfluence of a GPCR, e.g., a functional, physical, or chemical effect.It includes ligand binding, changes in ion flux, membrane potential,current flow, transcription, G-protein binding, gene amplification,expression in cancer cells, GPCR phosphorylation or dephosphorylation,signal transduction, receptor-ligand interactions, second messengerconcentrations (e.g., cAMP, cGMP, IP₃, DAG, or intracellular Ca²⁺), invitro, in vivo, and ex vivo and also includes other physiologic effectssuch as increases or decreases of neurotransmitter or hormone release;or increases in the synthesis of particular compounds, e.g.,triglycerides.

By “determining the functional effect” is meant assaying for a compoundthat increases or decreases a parameter that is indirectly or directlyunder the influence of a GPCR, e.g., functional, physical and chemicaleffects. Such functional effects can be measured by any means known tothose skilled in the art, e.g., changes in spectroscopic characteristics(e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g.,shape), chromatographic, or solubility properties, patch clamping,voltage-sensitive dyes, whole cell currents, radioisotope efflux,inducible markers, transcriptional activation of GPCRs; ligand bindingassays; voltage, membrane potential and conductance changes; ion fluxassays; changes in intracellular second messengers such as cAMP andinositol triphosphate (IP₃); changes in intracellular calcium levels;neurotransmitter release, and the like.

“Inhibitors,” “activators,” and “modulators” of GPCRs are usedinterchangeably to refer to inhibitory, activating, or modulatingmolecules identified using in vitro and in vivo assays for signaltransduction, e.g., ligands, agonists, antagonists, and their homologsand mimetics. Such modulating molecules, also referred to herein ascompounds, include polypeptides, antibodies, amino acids, nucleotides,lipids, carbohydrates, or any organic or inorganic molecule. Inhibitorsare compounds that, e.g., bind to, partially or totally blockstimulation, decrease, prevent, delay activation, inactivate,desensitize, or down regulate signal transduction, e.g., antagonists.Activators are compounds that, e.g., bind to, stimulate, increase, open,activate, facilitate, enhance activation, sensitize or up regulatesignal transduction, e.g., agonists. Modulators include compounds that,e.g., alter the interaction of a polypeptide with: extracellularproteins that bind activators or inhibitors; G-proteins; G-proteinalpha, beta, and gamma subunits; and kinases. Modulators also includegenetically modified versions of GPCRs, e.g., with altered activity, aswell as naturally occurring and synthetic ligands, antagonists,agonists, antibodies, small chemical molecules and the like. Such assaysfor inhibitors and activators include, e.g., expressing GPCRs in vitro,in cells or cell membranes, applying putative modulator compounds, andthen determining the functional effects on signal transduction, asdescribed above.

Samples or assays comprising GPCRs that are treated with a potentialactivator, inhibitor, or modulator are compared to control sampleswithout the inhibitor, activator, or modulator to examine the extent ofinhibition. Activation of a GPCR is achieved when the GPCR activityvalue relative to the control (untreated with activators) is 110%, morepreferably 150%, more preferably 200-500% (i.e., two to five fold higherrelative to the control), more preferably 1000-3000% higher. Fordetermining inhibitor activity, control samples (untreated withinhibitors) are assigned a relative GPCR activity value of 100%.Inhibition of a GPCR is achieved when the GPCR activity value relativeto the control is about 80%, preferably 50%, more preferably 25-0%.

The terms “isolated” “purified” or “biologically pure” refer to materialthat is substantially or essentially free from components which normallyaccompany it as found in its native state. Purity and homogeneity aretypically determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis or high performance liquidchromatography. A protein that is the predominant species present in apreparation is substantially purified. In particular, an isolated GPCRnucleic acid is separated from open reading frames that flank the GPCRgene and encode proteins other than the GPCR. The term “purified”denotes that a nucleic acid or protein gives rise to essentially oneband in an electrophoretic gel. Particularly, it means that the nucleicacid or protein is at least 85% pure, more preferably at least 95% pure,and most preferably at least 99% pure.

“Biologically active” GPCR refers to a GPCR having signal transductionactivity and G protein coupled receptor activity, as described above.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,and peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

-   1) Alanine (A), Glycine (G);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);-   7) Serine (S), Threonine (T); and-   8) Cysteine (C), Methionine (M)    (see, e.g., Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3^(rd) ed., 1994) and Cantor and Schimmel,Biophysical Chemistry Part I: The Conformation of BiologicalMacromolecules (1980). “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered, three dimensional structures within a polypeptide.These structures are commonly known as domains. Domains are portions ofa polypeptide that form a compact unit of the polypeptide and aretypically 25 to approximately 500 amino acids long. Typical domains aremade up of sections of lesser organization such as stretches of β-sheetand α-helices. “Tertiary structure” refers to the complete threedimensional structure of a polypeptide monomer. “Quaternary structure”refers to the three dimensional structure formed by the noncovalentassociation of independent tertiary units.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include ³²P, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, or haptens and proteins that can be madedetectable, e.g., by incorporating a radiolabel into the peptide, andused to detect antibodies specifically reactive with the peptide).

A “labeled nucleic acid probe or oligonucleotide” is one that is bound,either covalently, through a linker or a chemical bond, ornoncovalently, through ionic, van der Waals, electrostatic, or hydrogenbonds to a label such that the presence of the probe may be detected bydetecting the presence of the label bound to the probe.

As used herein a “nucleic acid probe or oligonucleotide” is defined as anucleic acid capable of binding to a target nucleic acid ofcomplementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. As used herein, a probe may include natural (i.e., A, G,C, or T) or modified bases (7-deazaguanosine, inosine, etc.). Inaddition, the bases in a probe may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere withhybridization. Thus, for example, probes may be peptide nucleic acids inwhich the constituent bases are joined by peptide bonds rather thanphosphodiester linkages. It will be understood by one of skill in theart that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are preferably directly labeledas with isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidin complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, or 95%identity over a specified region, when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using a BLAST or BLAST 2.0 sequence comparison algorithms withdefault parameters described below, or by manual alignment and visualinspection. Such sequences are then said to be “substantiallyidentical.” This definition also refers to the complement of a testsequence. Preferably, the identity exists over a region that is at leastabout 25 amino acids or nucleotides in length, or more preferably over aregion that is 50-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1994-1999).

A preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For selective or specific hybridization, apositive signal is at least two times background, optionally 10 timesbackground hybridization. Exemplary stringent hybridization conditionscan be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42°C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and0.1% SDS at 65° C. Such washes can be performed for 5, 15, 30, 60, 120,or more minutes. For PCR, a temperature of about 36° C. is typical forlow stringency amplification, although annealing temperatures may varybetween about 32° C. and 48° C. depending on primer length. For highstringency PCR amplification, a temperature of about 62° C. is typical,although high stringency annealing temperatures can range from about 50°C. to about 65° C., depending on the primer length and specificity.Typical cycle conditions for both high and low stringency amplificationsinclude a denaturation phase of 90° C.-95° C. for 30 sec-2 min., anannealing phase lasting 30 sec.-2 min., and an extension phase of about72° C. for 1-2 min.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay-libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

For preparation of monoclonal or polyclonal antibodies, any techniqueknown in the art can be used (see, e.g., Kohler & Milstein, Nature256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Coleet al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)).Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce antibodies to polypeptides of thisinvention. Also, transgenic mice, or other organisms such as othermammals, may be used to express humanized antibodies. Alternatively,phage display technology can be used to identify antibodies andheteromeric Fab fragments that specifically bind to selected antigens(see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al.,Biotechnology 10:779-783 (1992)).

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

An “anti-GPCR” antibody is an antibody or antibody fragment thatspecifically binds a polypeptide encoded by a GPCR gene, cDNA, or asubsequence thereof.

The term “immunoassay” is an assay that uses an antibody to specificallybind an antigen. The immunoassay is characterized by the use of specificbinding properties of a particular antibody to isolate, target, and/orquantify the antigen.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, polyclonal antibodiesraised to a particular GPCR can be selected to obtain only thosepolyclonal antibodies that are specifically immunoreactive with theGPCR, and not with other proteins, except for polymorphic variants,orthologs, and alleles of the GPCR. This selection may be achieved bysubtracting out antibodies that cross-react with GPCR molecules. Avariety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select antibodiesspecifically immunoreactive with a protein (see, e.g., Harlow & Lane,Antibodies, A Laboratory Manual (1988), for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity). Typically a specific or selective reaction will be atleast twice background signal or noise and more typically more than 10to 100 times background. Antibodies that react only with a particularGPCR ortholog, e.g., from specific species such as rat, mouse, or human,can also be made as described above, by subtracting out antibodies thatbind to the same GPCR from another species.

The phrase “selectively associates with” refers to the ability of anucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

Isolation of Nucleic Acids Encoding GPCRs

A. General Recombinant DNA Methods

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook & Russell, Molecular Cloning, A LaboratoryManual (3rd Ed, 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994-1999). Methods that are used to produceGPCRs for use in the invention may also be employed to produce proteinligands, e.g., stanniocalcins, ASP, or polypeptides that modulate ligandbinding to the receptor, for use in the invention.

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Protein sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862(1981), using an automated synthesizer, as described in Van Devanter et.al., Nucleic Acids Res. 12:6159-6168 (1984). Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson & Reanier, J. Chrom.255:137-149 (1983).

B. Cloning Methods for the Isolation of Nucleotide Sequences EncodingGPCRs

In general, the nucleic acid sequences encoding GPCRs and relatednucleic acid sequence homologs are cloned from cDNA and genomic DNAlibraries by hybridization with a probe, or isolated using amplificationtechniques with oligonucleotide primers, and verified by sequencing. Forexample, GPCR sequences are typically isolated from mammalian nucleicacid (genomic or cDNA) libraries by hybridizing with a nucleic acidprobe, the sequence of which can be derived from SEQ ID NO:1, 3, 5, 7,9, 11, 13, 15, or 17. Suitable tissues from which GPCR RNA and cDNA canbe isolated include, e.g., neural tissue, e.g., peripheral neural tissueand brain; immune cells and tissues, e.g., spleen, lymphocytes, bonemarrow, and the like; adipose tissue; bone tissue; and other tissues.

Amplification techniques using primers can also be used to amplify andisolate GPCR nucleic acids from DNA or RNA. Suitable primers can bedesigned using criteria well known in the art (see, e.g., Dieffenfach &Dveksler, PCR Primer: A Laboratory Manual (1995)). These primers can beused, e.g., to amplify either the full length sequence or a probe of oneto several hundred nucleotides, which is then used to screen a mammalianlibrary for full-length GPCRs.

Nucleic acids encoding GPCRs can also be isolated from expressionlibraries using antibodies as probes. Such polyclonal or monoclonalantibodies can be raised using the sequence of SEQ ID NO:2, 4, 6, 8, 10,12, 14, 16, or 18.

GPCR polymorphic variants, alleles, and interspecies homologs that aresubstantially identical to a GPCR can be isolated using GPCR nucleicacid probes, and oligonucleotides under stringent hybridizationconditions, by screening libraries. Alternatively, expression librariescan be used to clone GPCRs and GPCR polymorphic variants, alleles, andinterspecies homologs, by detecting expressed homologs immunologicallywith antisera or purified antibodies made against GPCRs, which alsorecognize and selectively bind to the GPCR homolog. Methods ofconstructing cDNA and genomic libraries are well known in the art (see,e.g., Sambrook & Russell, supra; and Ausubel et al., supra).

An alternative method of isolating GPCR nucleic acids and their homologscombines the use of synthetic oligonucleotide primers and amplificationof an RNA or DNA template (see U.S. Pat. Nos. 4,683,195 and 4,683,202;PCR Protocols: A Guide to Methods and Applications (Innis et al., eds,1990)). Methods such as polymerase chain reaction (PCR) and ligase chainreaction (LCR) can be used to amplify nucleic acid sequences of GPCRsdirectly from mRNA, from cDNA, from genomic libraries or cDNA libraries.Degenerate oligonucleotides can be designed to amplify GPCR homologsusing the sequences provided herein. Restriction endonuclease sites canbe incorporated into the primers. Polymerase chain reaction or other invitro amplification methods may also be useful, for example, to clonenucleic acid sequences that code for proteins to be expressed, to makenucleic acids to use as probes for detecting the presence ofGPCR-encoding mRNA in physiological samples, for nucleic acidsequencing, or for other purposes. Genes amplified by the PCR reactioncan be purified from agarose gels and cloned into an appropriate vector.

Gene expression can also be analyzed by techniques known in the art,e.g., reverse transcription and amplification of mRNA, isolation oftotal RNA or poly A⁺ RNA, northern blotting, dot blotting, in situhybridization, RNase protection, probing DNA microchip arrays, and thelike. In the case where the homologs being identified are linked to aknown disease, they can be used with GeneChip™ as a diagnostic tool indetecting the disease in a biological sample, see, e.g., Gunthand etal., AIDS Res. Hum. Retroviruses 14: 869-876 (1998); Kozal et al., Nat.Med. 2:753-759 (1996); Matson et al., Anal. Biochem. 224:110-106 (1995);Lockhart et al., Nat. Biotechnol. 14:1675-1680 (1996); Gingeras et al.,Genome Res. 8:435-448 (1998); Hacia et al., Nucleic Acids Res.26:3865-3866 (1998).

Synthetic oligonucleotides can be used to construct recombinant GPCRgenes for use as probes or for expression of protein. This method isperformed using a series of overlapping oligonucleotides usually 40-120bp in length, representing both the sense and nonsense strands of thegene. These DNA fragments are then annealed, ligated and cloned.Alternatively, amplification techniques can be used with precise primersto amplify a specific subsequence of the GPCR nucleic acid. The specificsubsequence is then ligated into an expression vector.

The nucleic acid encoding a GPCR is typically cloned into intermediatevectors before transformation into prokaryotic or eukaryotic cells forreplication and/or expression. These intermediate vectors are typicallyprokaryote vectors, e.g., plasmids, or shuttle vectors.

Optionally, nucleic acids encoding chimeric proteins comprising GPCRs ordomains thereof can be made according to standard techniques. Forexample, a domain such as ligand binding domain, an extracellulardomain, a transmembrane domain (e.g., one comprising seven transmembraneregions and corresponding extracellular and cytosolic loops), thetransmembrane domain and a cytoplasmic domain, an active site, a subunitassociation region, etc., can be covalently linked to a heterologousprotein. For example, an extracellular domain can be linked to aheterologous GPCR transmembrane domain, or a heterologous GPCRextracellular domain can be linked to a transmembrane domain. Otherheterologous proteins of choice include, e.g., green fluorescentprotein, luciferase, or β-gal.

C. Expression in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene or nucleic acid, suchas cDNAs encoding GPCRs, or a protein ligand, one typically subclones anucleic acid sequence encoding the protein of interest into anexpression vector that contains a strong promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook & Russell and Ausubel et al.Bacterial expression systems for expressing the protein are availablein, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983). Kits forsuch expression systems are commercially available. Eukaryoticexpression systems for mammalian cells, yeast, and insect cells are wellknown in the art and are also commercially available. In one embodiment,the eukaryotic expression vector is an adenoviral vector, anadeno-associated vector, or a retroviral vector.

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter is optionallypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the GPCR encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encoding a GPCRand signals required for efficient polyadenylation of the transcript,ribosome binding sites, and translation termination. The nucleic acidsequence encoding a GPCR may typically be linked to a cleavable signalpeptide sequence to promote secretion of the encoded protein by thetransformed cell. Such signal peptides would include, among others, thesignal peptides from tissue plasminogen activator, insulin, and neurongrowth factor, and juvenile hormone esterase of Heliothis virescens.Additional elements of the cassette may include enhancers and, ifgenomic DNA is used as the structural gene, introns with functionalsplice donor and acceptor sites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺,pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, polyhedrin promoter, orother promoters shown effective for expression in eukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. Alternatively, high yield expression systemsnot involving gene amplification are also suitable, such as using abaculovirus vector in insect cells, with a GPCR-encoding sequence underthe direction of the polyhedrin promoter or other strong baculoviruspromoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are optionally chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of GPCRprotein, which are then purified using standard techniques (see, e.g.,Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

Any of the well known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell (see,e.g., Russell & Sambrook, supra). It is only necessary that theparticular genetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressing aGPCR.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression of aGPCR, which is recovered from the culture using standard techniquesidentified below.

Transgenic animals, including knockout transgenic animals, that includeadditional copies of a GPCR and/or altered or mutated GPCR transgenescan also be generated. A “transgenic animal” refers to any animal (e.g.mouse, rat, pig, bird, or an amphibian), preferably a non-human mammal,in which one or more cells contain heterologous nucleic acid introducedusing transgenic techniques well known in the art. The nucleic acid isintroduced into the cell, directly or indirectly, by introduction into aprecursor of the cell, by way of deliberate genetic manipulation, suchas by microinjection or by infection with a recombinant virus. The termgenetic manipulation does not include classical cross-breeding, or invitro fertilization, but rather is directed to the introduction of arecombinant DNA molecule. This molecule may be integrated within achromosome, or it may be extrachromosomally replicating DNA.

In other embodiments, transgenic animals are produced in whichexpression of a GPCR is silenced. Gene knockout by homologousrecombination is a method that is commonly used to generate transgenicanimals. Transgenic mice can be derived using methodology known to thoseof skill in the art, see, e.g., Hogan et al., Manipulating the MouseEmbryo: A Laboratory Manual, (1988); Teratocarcinomas and Embryonic StemCells: A Practical Approach, Robertson, ed., (1987); and Capecchi etal., Science 244:1288 (1989).

Purification of GPCRs

Either naturally occurring or recombinant GPCRs can be purified for usein functional assays. Optionally, recombinant GPCRs are purified.Naturally occurring GPCRs are purified, e.g., from any suitable tissueor cell expressing naturally occurring GPCRs. Recombinant GPCRs arepurified from any suitable bacterial or eukaryotic expression system,e.g., CHO cells or insect cells.

A GPCR may be purified to substantial purity by standard techniques,including selective precipitation with such substances as ammoniumsulfate; column chromatography, immunopurification methods, and others(see, e.g., Scopes, Protein Purification: Principles and Practice(1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Russell &Sambrook, supra).

A number of procedures can be employed when a recombinant GPCR is beingpurified. For example, proteins having established molecular adhesionproperties can be reversibly fused to a GPCR. With the appropriateligand, a GPCR can be selectively adsorbed to a purification column andthen freed from the column in a relatively pure form. The fused proteinis then removed by enzymatic activity. Finally, a GPCR could be purifiedusing immunoaffinity columns.

Recombinant proteins are expressed by transformed bacteria or eukaryoticcells such as CHO cells or insect cells in large amounts, typicallyafter promoter induction; but expression can be constitutive. Promoterinduction with IPTG is one example of an inducible promoter system.Cells are grown according to standard procedures in the art. Fresh orfrozen cells are used for isolation of protein using techniques known inthe art (see, e.g., Russell & Sambrook, supra; and Ausubel et al.,supra).

Immunological Detection of GPCRs

In addition to the detection of GPCR genes and gene expression usingnucleic acid hybridization technology, one can also use antibodies todetect GPCRs that are used in the invention. A general overview of theapplicable technology can be found in Harlow & Lane, Antibodies: ALaboratory Manual (1988) and Harlow & Lane, Using Antibodies (1999).Again, these methods are also applicable to the preparation and use ofantibodies for other polypeptides used in this invention, e.g., ASP orstanniocalcin.

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with GPCRs are known to those of skill in the art (see,e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane,supra; Goding, Monoclonal Antibodies: Principles and Practice (2d ed.1986); and Kohler & Milstein, Nature 256:495-497 (1975). Such techniquesinclude antibody preparation by selection of antibodies from librariesof recombinant antibodies in phage or similar vectors, as well aspreparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989);Ward et al., Nature 341:544-546 (1989)). Such antibodies can be used fortherapeutic and diagnostic applications, e.g., in the treatment and/ordetection of any of the GPCR-associated diseases or conditions describedherein.

A number of GPCRs comprising immunogens may be used to produceantibodies specifically reactive with GPCRs. For example, a recombinantGPCR or an antigenic fragment thereof, is isolated as described herein.Recombinant protein can be expressed in eukaryotic or prokaryotic cellsas described above, and purified as generally described above.Recombinant protein is the preferred immunogen for the production ofmonoclonal or polyclonal antibodies. Alternatively, a synthetic peptidederived from the sequences disclosed herein and conjugated to a carrierprotein can be used as an immunogen. Naturally occurring protein mayalso be used either in pure or impure form. The product is then injectedinto an animal capable of producing antibodies. Either monoclonal orpolyclonal antibodies may be generated, for subsequent use inimmunoassays to measure the protein.

Typically, polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against non-GPCR proteinsor even other related proteins from other organisms, using a competitivebinding immunoassay. Specific polyclonal antisera and monoclonalantibodies will usually bind with a K_(d) of at least about 0.1 mM, moreusually at least about 1 μM, optionally at least about 0.1 μM or better,and optionally 0.01 μM or better.

Once GPCR specific antibodies are available, GPCRs can be detected by avariety of immunoassay methods. For a review of immunological andimmunoassay procedures, see Basic and Clinical Immunology (Stites & Terreds., 7th ed. 1991). Moreover, the immunoassays of the present inventioncan be performed in any of several configurations, which are reviewedextensively in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow &Lane, supra.

GPCRs can be detected and/or quantified using any of a number of wellrecognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Methods in Cell Biology: Antibodies inCell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology(Stites & Terr, eds., 7th ed. 1991). Immunological binding assays (orimmunoassays) typically use an antibody that specifically binds to aprotein or antigen of choice (in this case the GPCR or antigenicsubsequence thereof).

Immunoassays also often use a labeling agent to specifically bind to andlabel the complex formed by the antibody and antigen. The labeling agentmay itself be one of the moieties comprising the antibody/antigencomplex. Thus, the labeling agent may be a labeled GPCR polypeptide or alabeled anti-GPCR antibody. Alternatively, the labeling agent may be athird moiety, such as a secondary antibody, that specifically binds tothe antibody/GPCR complex (a secondary antibody is typically specific toantibodies of the species from which the first antibody is derived).Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelingagent. These proteins exhibit a strong non-immunogenic reactivity withimmunoglobulin constant regions from a variety of species (see, e.g.,Kronval et al., J. Immunol. 111:1401-1406 (1973); Akerstrom et al., J.Immunol. 135:2589-2542 (1985)). The labeling agent can be modified witha detectable moiety, such as biotin, to which another molecule canspecifically bind, such as streptavidin. A variety of detectablemoieties are well known to those skilled in the art.

Commonly used assays include noncompetitive assays, e.g., sandwichassays, and competitive assays. In competitive assays, the amount ofGPCR present in the sample is measured indirectly by measuring theamount of a known, added (exogenous) GPCR displaced (competed away) froman anti-GPCR antibody by the unknown GPCR present in a sample. Commonlyused assay formats include Western blots (immunoblots), which are usedto detect and quantify the presence of protein in a sample. Other assayformats include liposome immunoassays (LIA), which use liposomesdesigned to bind specific molecules (e.g., antibodies) and releaseencapsulated reagents or markers. The released chemicals are thendetected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:34-41 (1986)).

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecule (e.g., streptavidin), which iseither inherently detectable or covalently bound to a signal system,such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize GPCRs, orsecondary antibodies that recognize anti-GPCR.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidotases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

Cross-Reactivity Determinations

Immunoassays in the competitive binding format can also be used forcross-reactivity determinations. For example, a protein at leastpartially encoded by SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, or 17 can beimmobilized to a solid support. Proteins (e.g., GPCR proteins andhomologs) are added to the assay that compete for binding of theantisera to the immobilized antigen. The ability of the added proteinsto compete for binding of the antisera to the immobilized protein iscompared to the ability of GPCRs encoded by SEQ ID NO:1, 3, 5, 7, 9, 11,13, 15, or 17 to compete with itself. The percent crossreactivity forthe above proteins is calculated, using standard calculations. Thoseantisera with less than 10% crossreactivity with each of the addedproteins listed above are selected and pooled. The cross-reactingantibodies are optionally removed from the pooled antisera byimmunoabsorption with the added considered proteins, e.g., distantlyrelated homologs.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps an allele or polymorphic variant of a GPCR, to theimmunogen protein (i.e., the GPCR of SEQ ID NO:2, 4, 6, 8, 10, 12, 14,16, or 18). In order to make this comparison, the two proteins are eachassayed at a wide range of concentrations and the amount of each proteinrequired to inhibit 50% of the binding of the antisera to theimmobilized protein is determined. If the amount of the second proteinrequired to inhibit 50% of binding is less than 10 times the amount ofthe protein encoded by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17 thatis required to inhibit 50% of binding, then the second protein is saidto specifically bind to the polyclonal antibodies generated to a GPCRimmunogen.

Assays for Modulators of GPCRs

A. Assays for GPCR Activity

GPCRs and their alleles and polymorphic variants are G-protein coupledreceptors that participate in signal transduction and are associatedwith cellular function in a variety of cells, e.g., neurons, immunesystem cells, kidney, liver, colon, adipose, and other cells. Theactivity of GPCR polypeptides can be assessed using a variety of invitro and in vivo assays to determine functional, chemical, and physicaleffects, e.g., measuring ligand binding, (e.g., radioactive ligandbinding), second messengers (e.g., cAMP, cGMP, IP₃, DAG, or Ca²⁺), ionflux, phosphorylation levels, transcription levels, neurotransmitterlevels, and the like. Such assays can be used to test for inhibitors andactivators of a GPCR. In particular, the assays can be used to test forcompounds that modulate natural ligand-induced GPCR activity, forexample, by modulating the binding of the natural ligand to the receptorand/or by modulating the ability of the natural ligand to activate thereceptor. Typically in such assays, the test compound is contacted withthe GPCR in the presence of the natural ligand. The natural ligand maybe added to the assay before, after, or concurrently with the testcompound. The results of the assay, for example, the level of binding,calcium mobilization, etc. is then compared to the level in a controlassay that comprises the GPCR and natural ligand in the absence of thetest compound.

Screening assays of the invention are used to identify modulators thatcan be used as therapeutic agents, e.g., antibodies to GPCRs andantagonists of GPCR activity.

The effects of test compounds upon the function of the GPCR polypeptidescan be measured by examining any of the parameters described above. Anysuitable physiological change that affects GPCR activity can be used toassess the influence of a test compound on the GPCRs and naturalligand-mediated GPCR activity. When the functional consequences aredetermined using intact cells or animals, one can also measure a varietyof effects such as transmitter release, hormone release, transcriptionalchanges to both known and uncharacterized genetic markers (e.g.,northern blots), changes in cell metabolism such as cell growth or pHchanges, and changes in intracellular second messengers such as Ca²⁺,IP₃ or cAMP.

For a general review of GPCR signal transduction and methods of assayingsignal transduction, see, e.g., Methods in Enzymology, vols. 237 and 238(1994) and volume 96 (1983); Bourne et al., Nature 10:349:117-27 (1991);Bourne et al., Nature 348:125-32 (1990); Pitcher et al., Annu. Rev.Biochem. 67:653-92 (1998).

The GPCR of the assay will be selected from a polypeptide having asequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, or 18, orconservatively modified variants thereof. Alternatively, the GPCR of theassay will be derived from a eukaryote and include an amino acidsubsequence having amino acid sequence identity to SEQ ID NO:2, 4, 6, 8,10, 12 14, 16 or 18. Generally, the amino acid sequence identity will beat least 70%, optionally at least 80%, optionally at least 90-95%.Optionally, the polypeptide of the assays will comprise or consist of adomain of a GPCR, such as an extracellular domain, transmembrane domain,cytoplasmic domain, ligand binding domain, subunit association domain,active site, and the like. Either a GPCR or a domain thereof can becovalently linked to a heterologous protein to create a chimeric proteinused in the assays described herein.

Modulators of GPCR activity are tested using GPCR polypeptides asdescribed above, either recombinant or naturally occurring. The proteincan be isolated, expressed in a cell, expressed in a membrane derivedfrom a cell, expressed in tissue or in an animal, either recombinant ornaturally occurring. For example, neurons, cells of the immune system,adipocytes, kidney cells, transformed cells, or membranes can be used.Modulation is tested using one of the in vitro or in vivo assaysdescribed herein. Signal transduction can also be examined in vitro withsoluble or solid state reactions, using a chimeric molecule such as anextracellular domain of a receptor covalently linked to a heterologoussignal transduction domain, or a heterologous extracellular domaincovalently linked to the transmembrane and or cytoplasmic domain of areceptor. Furthermore, ligand-binding domains of the protein of interestcan be used in vitro in soluble or solid state reactions to assay forligand binding.

Ligand binding to a GPCR, a domain, or chimeric protein can be tested ina number of formats. For example, binding can be performed in solution,in a bilayer membrane, attached to a solid phase, in a lipid monolayer,or in vesicles. Typically, in an assay of the invention, the binding ofthe natural ligand to its receptor is measured in the presence of acandidate modulator. Alternatively, the binding of the candidatemodulator may be measured in the presence of the natural ligand. Often,competitive assay that measure the ability of a compound to compete withbinding of the natural ligand to the receptor are used. Binding may bemeasured by assessing GPCR activity or by other assays: binding can betested by measuring e.g., changes in spectroscopic characteristics(e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g.,shape) changes, or changes in chromatographic or solubility properties.

Receptor-G-protein interactions can also be used to assay formodulators. For example, in the absence of GTP, binding of an activatorsuch as the natural ligand will lead to the formation of a tight complexof a G protein (all three subunits) with the receptor. This complex canbe detected in a variety of ways, as noted above. Such an assay can bemodified to search for inhibitors. For example, the ligand can be addedto the receptor and G protein in the absence of GTP to form a tightcomplex. Inhibitors may be identified by looking at dissociation of thereceptor-G protein complex. In the presence of GTP, release of the alphasubunit of the G protein from the other two G protein subunits serves asa criterion of activation.

An activated or inhibited G-protein will in turn alter the properties ofdownstream effectors such as proteins, enzymes, and channels. Theclassic examples are the activation of cGMP phosphodiesterase bytransducin in the visual system, adenylate cyclase by the stimulatoryG-protein, phospholipase C by G_(q) and other cognate G proteins, andmodulation of diverse channels by Gi and other G proteins. Downstreamconsequences such as generation of diacyl glycerol and IP₃ byphospholipase C, and in turn, for calcium mobilization e.g., by IP₃(further discussed below) can also be examined. Thus, modulators can beevaluated for the ability to stimulate or inhibit ligand-mediateddownstream effects. For example, β-alanine specifically activates TGR2in inositol phosphate accumulation and calcium mobilization, e.g.,aequorin, assays. Similarly, GPR40 and GPR43 are activated in aequorinassays when bound by the fatty acid ligands disclosed herein; and TGR18and TGR164 are activated by short chain carboxylic acids in calciummobilization assays and inositol phosphate assays. Candidate modulatorsmay be assessed for the ability to inhibit inositol phosphateaccumulation and/or calcium mobilization induced by the natural ligand,e.g., β-alanine (TGR2), a 2-3 carbon chain fatty acid (GPR43), a mediumor long chain fatty acid (GPR40), succinic acid (TGR18), ora-ketoglutaric acid (TGR164).

In other examples, the ability of a modulator to activate a GPRexpressed in adipocytes, e.g., GPR43, in comparison to the ability of anatural ligand fatty acid, may be determined using assays such aslipolysis (see, e.g., WO01/61359).

Activated GPCRs become substrates for kinases that phosphorylate theC-terminal tail of the receptor (and possibly other sites as well).Thus, activators will promote the transfer of ³²P from gamma-labeled GTPto the receptor, which can be assayed with a scintillation counter. Thephosphorylation of the C-terminal tail will promote the binding ofarrestin-like proteins and will interfere with the binding ofG-proteins. The kinase/arrestin pathway plays a key role in thedesensitization of many GPCR receptors.

Modulators may therefore also be identified using assays involvingβ-arrestin recruitment. β-arrestin serves as a regulatory protein thatis distributed throughout the cytoplasm in unactivated cells. Ligandbinding to an appropriate GPCR is associated with redistribution ofβ-arrestin from the cytoplasm to the cell surface, where it associateswith the GPCR. Thus, receptor activation and the effect of candidatemodulators on ligand-induced receptor activation, can be assessed bymonitoring β-arrestin recruitment to the cell surface. This isfrequently performed by transfecting a labeled β-arrestin fusion protein(e.g., β-arrestin-green fluorescent protein (GFP)) into cells andmonitoring its distribution using confocal microscopy (see, e.g.,Groarke et al., J. Biol. Chem. 274(33):23263-69 (1999)).

Receptor internalization assays may also be used to assess receptorfunction. Upon ligand binding, the G-protein coupled receptor—ligandcomplex is internalized from the plasma membrane by a clathrin-coatedvesicular endocytic process; internalization motifs on the receptorsbind to adaptor protein complexes and mediate the recruitment of theactivated receptors into clathrin-coated pits and vesicles. Because onlyactivated receptors are internalized, it is possible to detectligand-receptor binding by determining the amount of internalizedreceptor. In one assay format, cells are transiently transfected withradiolabeled receptor and incubated for an appropriate period of time toallow for ligand binding and receptor internalization. Thereafter,surface-bound radioactivity is removed by washing with an acid solution,the cells are solubilized, and the amount of internalized radioactivityis calculated as a percentage of ligand binding. See, e.g., Vrecl etal., Mol. Endocrinol. 12:1818-29 (1988) and Conway et al., J. CellPhysiol. 189(3):341-55 (2001). In addition, receptor internalizationapproaches have allowed real-time optical measurements of GPCRinteractions with other cellular components in living cells (see, e.g.,Barak et al., Mol. Pharmacol. 51(2)177-84 (1997)). Modulators may beidentified by comparing receptor internalization levels in control cellsand cells contacted with candidate compounds. For example, candidatemodulators are assayed by examining their effects on receptorinternalization upon binding of the natural ligand, β-alanine, ASP,stanniocalcin, a medium or long chain fatty acid, or a 2-3 carbon chainfatty acid, succinic acid, or α-ketoglutaric acid to its cognatereceptor, i.e., TGR2; GPR77; or LGR4, LGR5, LGR6, GPR40, GPR43, TGR18,or TGR164; respectively.

Another technology that can be used to evaluate GPCR-proteininteractions in living cells involves bioluminescence resonance energytransfer (BRET). A detailed discussion regarding BRET can be found inKroeger et al., J. Biol. Chem., 276(16):12736-43 (2001).

Receptor-stimulated guanosine 5′-O-(γ-Thio)-Triphosphate ([³⁵S]GTPγS)binding to G-proteins may also be used as an assay for evaluatingmodulators of GPCRs. [³⁵S]GTPγS is a radiolabeled GTP analog that has ahigh affinity for all types of G-proteins, is available with a highspecific activity and, although unstable in the unbound form, is nothydrolyzed when bound to the G-protein. Thus, it is possible toquantitatively assess ligand-bound receptor by comparing stimulatedversus unstimulated [³⁵S]GTPγS binding utilizing, for example, a liquidscintillation counter. Inhibitors of the receptor-ligand interactionswould result in decreased [³⁵S]GTPγS binding. Descriptions of [³⁵S]GTPγSbinding assays are provided in Traynor and Nahorski, Mol. Pharmacol.47(4):848-54 (1995) and Bohn et al., Nature 408:720-23 (2000).

The ability of modulators to affect ligand-induced ion flux may also bedetermined. Ion flux may be assessed by determining changes inpolarization (i.e., electrical potential) of the cell or membraneexpressing a GPCR. One means to determine changes in cellularpolarization is by measuring changes in current (thereby measuringchanges in polarization) with voltage-clamp and patch-clamp techniques,e.g., the “cell-attached” mode, the “inside-out” mode, and the “wholecell” mode (see, e.g., Ackerman et al., New Engl. J. Med. 336:1575-1595(1997)). Whole cell currents are conveniently determined using thestandard methodology (see, e.g., Hamil et al., PFlugers. Archiv. 391:85(1981). Other known assays include: radiolabeled ion flux assays andfluorescence assays using voltage-sensitive dyes (see, e.g.,Vestergarrd-Bogind et al., J. Membrane Biol. 88:67-75 (1988); Gonzales &Tsien, Chem. Biol. 4:269-277 (1997); Daniel et al., J. Pharmacol. Meth.25:185-193 (1991); Holevinsky et al., J. Membrane Biology 137:59-70(1994)). Generally, the compounds to be tested are present in the rangefrom 1 pM to 100 mM.

Preferred assays for G-protein coupled receptors include cells that areloaded with ion or voltage sensitive dyes to report receptor activity.Assays for determining activity of such receptors can also use knownagonists and antagonists for other G-protein coupled receptors and thenatural ligands disclosed herein as negative or positive controls toassess activity of tested compounds. In assays for identifyingmodulatory compounds (e.g., agonists, antagonists), changes in the levelof ions in the cytoplasm or membrane voltage are monitored using an ionsensitive or membrane voltage fluorescent indicator, respectively. Amongthe ion-sensitive indicators and voltage probes that may be employed arethose disclosed in the Molecular Probes 1997 Catalog. For G-proteincoupled receptors, promiscuous G-proteins such as Gα15 and Gα16 can beused in the assay of choice (Wilkie et al., Proc. Nat'l Acad. Sci. USA88:10049-10053 (1991)). Such promiscuous G-proteins allow coupling of awide range of receptors to signal transduction pathways in heterologouscells.

As noted above, receptor activation by ligand binding typicallyinitiates subsequent intracellular events, e.g., increases in secondmessengers such as IP₃, which releases intracellular stores of calciumions. Activation of some G-protein coupled receptors stimulates theformation of inositol triphosphate (IP₃) through phospholipaseC-mediated hydrolysis of phosphatidylinositol (Berridge & Irvine, Nature312:315-21 (1984)). IP₃ in turn stimulates the release of intracellularcalcium ion stores. Thus, a change in cytoplasmic calcium ion levels, ora change in second messenger levels such as IP₃ can be used to assessG-protein coupled receptor function. Cells expressing such G-proteincoupled receptors may exhibit increased cytoplasmic calcium levels as aresult of contribution from both intracellular stores and via activationof ion channels, in which case it may be desirable although notnecessary to conduct such assays in calcium-free buffer, optionallysupplemented with a chelating agent such as EGTA, to distinguishfluorescence response resulting from calcium release from internalstores.

Other assays can involve determining the activity of receptors which,when activated by ligand binding, result in a change in the level ofintracellular cyclic nucleotides, e.g., cAMP or cGMP, by activating orinhibiting downstream effectors such as adenylate cyclase. There arecyclic nucleotide-gated ion channels, e.g., rod photoreceptor cellchannels and olfactory neuron channels that are permeable to cationsupon activation by binding of cAMP or cGMP (see, e.g., Altenhofen etal., Proc. Natl. Acad. Sci. U.S.A. 88:9868-9872 (1991) and Dhallan etal., Nature 347:184-187 (1990)). In cases where activation of thereceptor results in a decrease in cyclic nucleotide levels, it may bepreferable to expose the cells to agents that increase intracellularcyclic nucleotide levels, e.g., forskolin, prior to adding areceptor-activating compound to the cells in the assay. Cells for thistype of assay can be made by co-transfection of a host cell with DNAencoding a cyclic nucleotide-gated ion channel, GPCR phosphatase and DNAencoding a receptor (e.g., certain glutamate receptors, muscarinicacetylcholine receptors, dopamine receptors, serotonin receptors, andthe like), which, when activated, causes a change in cyclic nucleotidelevels in the cytoplasm.

In one embodiment, changes in intracellular cAMP or cGMP can be measuredusing immunoassays. The method described in Offermanns & Simon, J. Biol.Chem. 270:15175-15180 (1995) may be used to determine the level of cAMP.Also, the method described in Felley-Bosco et al., Am. J. Resp. Cell andMol. Biol. 11:159-164 (1994) may be used to determine the level of cGMP.Further, an assay kit for measuring cAMP and/or cGMP is described inU.S. Pat. No. 4,115,538, herein incorporated by reference.

In another embodiment, phosphatidyl inositol (PI) hydrolysis can beanalyzed according to U.S. Pat. No. 5,436,128, herein incorporated byreference. Briefly, the assay involves labeling of cells with³H-myoinositol for 48 or more hrs. The labeled cells are treated with atest compound for one hour. The treated cells are lysed and extracted inchloroform-methanol-water after which the inositol phosphates areseparated by ion exchange chromatography and quantified by scintillationcounting. Fold stimulation is determined by calculating the ratio of cpmin the presence of agonist to cpm in the presence of buffer control.Likewise, fold inhibition is determined by calculating the ratio of cpmin the presence of antagonist to cpm in the presence of buffer control(which may or may not contain an agonist).

In another embodiment, transcription levels can be measured to assessthe effects of a test compound on ligand-induced signal transduction. Ahost cell containing the protein of interest is contacted with a testcompound in the presence of the natural ligand for a sufficient time toeffect any interactions, and then the level of gene expression ismeasured. The amount of time to effect such interactions may beempirically determined, such as by running a time course and measuringthe level of transcription as a function of time. The amount oftranscription may be measured by using any method known to those ofskill in the art to be suitable. For example, mRNA expression of theprotein of interest may be detected using northern blots or theirpolypeptide products may be identified using immunoassays.Alternatively, transcription based assays using reporter genes may beused as described in U.S. Pat. No. 5,436,128, herein incorporated byreference. The reporter genes can be, e.g., chloramphenicolacetyltransferase, firefly luciferase, bacterial luciferase,β-galactosidase and alkaline phosphatase. Furthermore, the protein ofinterest can be used as an indirect reporter via attachment to a secondreporter such as green fluorescent protein (see, e.g., Mistili &Spector, Nature Biotechnology 15:961-964 (1997)).

The amount of transcription is then compared to the amount oftranscription in either the same cell in the absence of the testcompound, or it may be compared with the amount of transcription in asubstantially identical cell that lacks the protein of interest. Asubstantially identical cell may be derived from the same cells fromwhich the recombinant cell was prepared but which had not been modifiedby introduction of heterologous DNA. Any difference in the amount oftranscription indicates that the test compound has in some manneraltered the activity of the protein of interest.

Samples that are treated-with a potential GPCR inhibitor or activatorare compared to control samples comprising the natural ligand withoutthe test compound to examine the extent of modulation. Control samples(untreated with activators or inhibitors) are assigned a relative GPCRactivity value of 100. Inhibition of a GPCR is achieved when the GPCRactivity value relative to the control is about 90%, optionally 50%,optionally 25-0%. Activation of a GPCR is achieved when the GPCRactivity value relative to the control is 110%, optionally 150%,200-500%, or 1000-2000%.

B. Modulators

The compounds tested as modulators of GPCRs can be any small chemicalcompound, or a biological entity, e.g., a macromolecule such as aprotein, sugar, nucleic acid or lipid. Alternatively, modulators can begenetically altered versions of a GPCR. Typically, test compounds willbe small chemical molecules and peptides. Essentially any chemicalcompound can be used as a potential modulator or ligand in the assays ofthe invention, although most often compounds can be dissolved in aqueousor organic (especially DMSO-based) solutions. The assays are designed toscreen large chemical libraries by automating the assay steps. Theassays are typically run in parallel (e.g., in microtiter formats onmicrotiter plates in robotic assays). It will be appreciated that thereare many suppliers of chemical compounds, including Sigma (St. Louis,Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), FlukaChemika-Biochemica Analytika (Buchs Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091),benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat.Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagiharaet al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Russell & Sambrook, all supra),peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083),antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology,14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see,e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S. Pat. No.5,593,853), small organic molecule libraries (see, e.g.,benzodiazepines, Baum C&EN, January 18, page 33 (1993); isoprenoids,U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat.No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;morpholino compounds, U.S. Pat. Nos. 5,506,337; benzodiazepines,5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, Mo., 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

C. Solid State and Soluble High Throughput Assays

In one embodiment the invention provides soluble assays using moleculessuch as a domain, e.g., a ligand binding domain, an extracellulardomain, a transmembrane domain (e.g., one comprising seven transmembraneregions and cytosolic loops), the transmembrane domain and a cytoplasmicdomain, an active site, a subunit association region, etc.; a domainthat is covalently linked to a heterologous protein to create a chimericmolecule; a GPCR; or a cell or tissue expressing a GPCR, eithernaturally occurring or recombinant. In another embodiment, the inventionprovides solid phase based in vitro assays in a high throughput format,where the domain, chimeric molecule, GPCR, or cell or tissue expressinga GPCR is attached to a solid phase substrate.

In the high throughput assays of the invention, it is possible to screenup to several thousand different modulators or ligands in a single day.In particular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100-1500 differentcompounds. It is possible to assay several different plates per day;assay screens for up to about 6,000-20,000 different compounds ispossible using the integrated systems of the invention.

The molecule of interest can be bound to the solid state component,directly or indirectly, via covalent or non covalent linkage e.g., via atag. The tag can be any of a variety of components. In general, amolecule which binds the tag (a tag binder) is fixed to a solid support,and the tagged molecule of interest (e.g., the signal transductionmolecule of interest) is attached to the solid support by interaction ofthe tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.).Antibodies to molecules with natural binders such as biotin are alsowidely available and are appropriate tag binders; see, SIGMAImmunochemicals 1998 catalogue SIGMA, St. Louis Mo.).

Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Thousands ofspecific antibodies are commercially available and many additionalantibodies are described in the literature. For example, in one commonconfiguration, the tag is a first antibody and the tag binder is asecond antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs. For example, agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherein family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g. which mediate the effects of various smallligands, including steroids, thyroid hormone, retinoids and vitamin D;peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclicpolymer configurations), oligosaccharides, proteins, phospholipids andantibodies can all interact with various cell receptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, and polyacetates can also form an appropriatetag or tag binder. Many other tag/tag binder pairs are also useful inassay systems described herein, as would be apparent to one of skillupon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences, such as poly-gly sequencesof between about 5 and 200 amino acids. Such flexible linkers are knownto persons of skill in the art. For example, poly(ethelyne glycol)linkers are available from Shearwater Polymers, Inc. Huntsville, Ala.These linkers optionally have amide linkages, sulfhydryl linkages, orheterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature. See, e.g., Merrifield, J.Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)(describing synthesis of solid phase components on pins); Frank &Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al., Science,251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719(1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (alldescribing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

D. Computer-Based Assays

Yet another assay for compounds that modulate GPCR activity involvescomputer assisted drug design, in which a computer system is used togenerate a three-dimensional structure of GPCR based on the structuralinformation encoded by the amino acid sequence. The input amino acidsequence interacts directly and actively with a preestablished algorithmin a computer program to yield secondary, tertiary, and quaternarystructural models of the protein. The models of the protein structureare then examined to identify the regions that have the ability to bind,e.g., ligands. These regions are then used to identify various compoundsthat modulate ligand-receptor binding.

The three-dimensional structural model of the protein is generated byentering protein amino acid sequences of at least 10 amino acid residuesor corresponding nucleic acid sequences encoding a GPCR polypeptide intothe computer system. The amino acid sequence of the polypeptide or thenucleic acid encoding the polypeptide is selected from the groupconsisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18; or SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, or 17, respectively, and conservativelymodified versions thereof. The amino acid sequence represents theprimary sequence or subsequence of the protein, which encodes thestructural information of the protein. At least 10 residues of the aminoacid sequence (or a nucleotide sequence encoding 10 amino acids) areentered into the computer system from computer keyboards, computerreadable substrates that include, but are not limited to, electronicstorage media (e.g., magnetic diskettes, tapes, cartridges, and chips),optical media (e.g., CD ROM), information distributed by internet sites,and by RAM. The three-dimensional structural model of the protein isthen generated by the interaction of the amino acid sequence and thecomputer system, using software known to those of skill in the art.

The amino acid sequence represents a primary structure that encodes theinformation necessary to form the secondary, tertiary and quaternarystructure of the protein of interest. The software looks at certainparameters encoded by the primary sequence to generate the structuralmodel. These parameters are referred to as “energy terms,” and primarilyinclude electrostatic potentials, hydrophobic potentials, solventaccessible surfaces, and hydrogen bonding. Secondary energy termsinclude van der Waals potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel.

The tertiary structure of the protein encoded by the secondary structureis then formed on the basis of the energy terms of the secondarystructure. The user at this point can enter additional variables such aswhether the protein is membrane bound or soluble, its location in thebody, and its cellular location, e.g., cytoplasmic, surface, or nuclear.These variables along with the energy terms of the secondary structureare used to form the model of the tertiary structure. In modeling thetertiary structure, the computer program matches hydrophobic faces ofsecondary structure with like, and hydrophilic faces of secondarystructure with like.

Once the structure has been generated, potential ligand binding regionsare identified by the computer system. Three-dimensional structures forpotential ligands are generated by entering amino acid or nucleotidesequences or chemical formulas of compounds, as described above. Thethree-dimensional structure of the potential ligand is then compared tothat of the GPCR protein to identify ligands that bind to GPCR. Bindingaffinity between the protein and ligands is determined using energyterms to determine which ligands have an enhanced probability of bindingto the protein.

Computer systems are also used to screen for mutations, polymorphicvariants, alleles and interspecies homologs of GPCR genes. Suchmutations can be associated with disease states or genetic traits. Asdescribed above, GeneChip™ and related technology can also be used toscreen for mutations, polymorphic variants, alleles and interspecieshomologs. Once the variants are identified, diagnostic assays can beused to identify patients having such mutated genes. Identification ofthe mutated GPCR genes involves receiving input of a first nucleic acidor amino acid sequence encoding an GPCR, selected from the groupconsisting of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, or 17; or SEQ IDNO:2, 4, 6, 8, 10, 12, 14, 16, or 18, respectively, and conservativelymodified versions thereof. The sequence is entered into the computersystem as described above. The first nucleic acid or amino acid sequenceis then compared to a second nucleic acid or amino acid sequence thathas substantial identity to the first sequence. The second sequence isentered into the computer system in the manner described above. Once thefirst and second sequences are compared, nucleotide or amino aciddifferences between the sequences are identified. Such sequences canrepresent allelic differences in GPCR genes, and mutations associatedwith disease states and genetic traits.

E. Expression Assays

Certain screening methods involve screening for a compound that modulatethe expression of the GPCRs described herein, or the levels of naturalligands, e.g., ASP and stanniocalcins. Such methods generally involveconducting cell-based assays in which test compounds are contacted withone or more cells expressing the GPCR or ligand and then detecting anincrease or decrease in expression (either transcript or translationproduct). Such assays are typically performed with cells that expressthe endogenous GPCR or ligand.

Expression can be detected in a number of different ways. As describedherein, the expression levels of the protein in a cell can be determinedby probing the mRNA expressed in a cell with a probe that specificallyhybridizes with a transcript (or complementary nucleic acid derivedtherefrom) of the GPCR or protein ligand. Probing can be conducted bylysing the cells and conducting Northern blots or without lysing thecells using in situ-hybridization techniques (see above). Alternatively,protein can be detected using immunological methods in which a celllysate is probed with antibodies that specifically bind to the protein.

Other cell-based assays are reporter assays conducted with cells that donot express the protein. Certain of these assays are conducted with aheterologous nucleic acid construct that includes a promoter that isoperably linked to a reporter gene that encodes a detectable product. Anumber of different reporter genes can be utilized. Some reporters areinherently detectable. An example of such a reporter is greenfluorescent protein that emits fluorescence that can be detected with afluorescence detector. Other reporters generate a detectable product.Often such reporters are enzymes. Exemplary enzyme reporters include,but are not limited to, β-glucuronidase, CAT (chloramphenicol acetyltransferase), luciferase, β-galactosidase and alkaline phosphatase.

In these assays, cells harboring the reporter construct are contactedwith a test compound. A test compound that either modulates the activityof the promoter by binding to it or triggers a cascade that produces amolecule that modulates the promoter causes expression of the detectablereporter. Certain other reporter assays are conducted with cells thatharbor a heterologous construct that includes a transcriptional controlelement that activates expression of the GPCR or ligand and a reporteroperably linked thereto. Here, too, an agent that binds to thetranscriptional control element to activate expression of the reporteror that triggers the formation of an agent that binds to thetranscriptional control element to activate reporter expression, can beidentified by the generation of signal associated with reporterexpression.

Kits

GPCRs and their homologs are a useful tool for identifying cells such asimmune cells, adipose, or neural cells, for forensics and paternitydeterminations, for diagnosing diseases, and for examining signaltransduction. GPCR-specific reagents that specifically bind to a GPCRprotein, e.g., the ligand, or GPCR antibodies are used to examine signaltransduction regulation.

The present invention also provides for kits for screening formodulators of ligand-GPCR interactions. Such kits can be prepared fromreadily available materials and reagents. For example, such kits cancomprise any one or more of the following materials: a GPCR, reactiontubes, and instructions for testing GPCR activity. Optionally, the kitcontains biologically active GPCR. A wide variety of kits and componentscan be prepared according to the present invention, depending upon theintended user of the kit and the particular needs of the user.

Disease Treatment and Diagnosis

TGRs are involved in the regulation of many important physiologicalfunctions and are often therapeutic targets for various diseases orconditions. Mammalian TGRs are typically classified in three categories,class A, receptors related to rhodopsin and the adrenergic receptors,class B, receptors related to the calcitonin and parathyroid hormonereceptors, and class C, receptors related to the metabotropic receptors.The rhodopsin/adrenergic receptor class is the largest class andincludes various amine receptor, e.g., acetylcholine (muscarinic)receptors, adrenergic receptors, dopamine receptors, histaminereceptors, serotonin receptors, and octopamine receptors; peptidereceptors, e.g., angiotensin, bombesin, bradykinin, endothelin,interleukin-8, chemokine, melanocortin, neuropeptide Y, neurotensin,opioid, somatostatin, tachykinin, thrombin, vasopressin, galanin,proteinase-activated, orexin, and chemokine/chemotatic factor receptors;protein hormone receptors, e.g., FSH, lutropin-choriogonadotropichormone, and thyrotropin receptors; rhodopsin receptors; olfactoryreceptors; prostanoid receptors; nucleotide-like receptors, includingadenosine and purinoceptors; cannabis receptors; platelet activatingfactor receptor; gonadotropin-releasing hormone receptor; melatoninreceptor, lysosphingolipid and LPA (EDG) receptors, as well as variousorphan receptors. Class B includes calcitonin, corticotropin releasingfactor, gastric inhibitory peptide glucagon, growth hormone-releasinghormone, parathyroid hormone, PACAP, secretin, vasoactive intestinalpolypeptide, and brain-specific angiogenesis inhibitor receptors, amongothers. Class C receptors include metabotropic glutamate receptors andGABA-B subtype receptors as well as putative pheromone receptors.

Class A GPCRs function in a variety of physiological processes such asvasodilation, bronchodilation, neurotransmitter signaling, stimulationof endocrine secretions, gut peristalsis, development, mitogenesis, cellproliferation, cell migration, immune system function, and oncogenesis.Accordingly, class A GPCRs can be used, for example, as probes toidentify cells or tissues that exhibit dysregulation of these processes,and moreover, as screening targets to identify modulators of theseprocesses.

Class B GPCRs also function in a wide range of physiological processessuch as regulation of calcium homeostasis, modulation of activity ofcells in the immune system, various excitatory and inhibitory actions inthe central nervous system, control of smooth muscle relaxation, controlof smooth muscle, secretion in stomach, intestinal epithelium, pancreas,and gall bladder. Accordingly, class B GPCRs can be used, for example,as probes to identify cells or tissues that exhibit dyregulation ofthese process, and to identify modulators of these physiologicalprocesses.

Class C GPCRs, metabotropic glutamate receptors, are also importantregulators of physiological processes such as neurotransmission.Glutamate is the major neurotransmitter in the CNS and plays animportant role in neuronal plasticity, cognition, memory, learning, andsome neurological disorders such as epilepsy, stroke, andneurodegeneration. B-type receptors for the neurotransmitter GABA(gamma-aminobutyric acid) inhibit neuronal activity throughG-protein-coupled second-messenger systems, which regulate the releaseof neurotransmitters and the activity of ion channels and adenylylcyclase. Thus, GABA B-type receptors play a role in controlling neuronalfunction and are also involved in such processes as neuronal plasticity,cognition, memory, and learning. Accordingly, class C GPCRs can be used,for example, as probes to identify cells or tissues, particularly,neuronal cells or tissues, that exhibit dysregulation of theseprocesses, and to identify modulators of these physiological processesfor the treatment of neurological disorders.

In certain embodiments, the presently-described GPCRs can be used in thediagnosis and treatment of certain diseases or conditions, i.e.,TGR-associated disorders. For example, modulators of the activity ofGPCRs (e.g., TGR2, GPR77, LGR4, LGR5, LGR6, GPR40, GPR43, TGR18, orTGR164) that are expressed in a particular cell type (e.g., a peripheralneuron, immune cell, brain tissue, adipocytes, kidney cells), can beused to modulate cellular function and pathways that involve that celltype (e.g., responsiveness to extracellular signals, such as pain, orinflammatory signals; or metabolic pathways, such as lipid metabolism).Thus, modulators may be used to treat conditions or diseases with TGR2,GPR77, LGR4, LGR5, LGR6, GPR40, GPR43, TGR18, or TGR164. For example,modulators of GPR40 or GPR43 activity may be used to treat diseasesassociated with lipid metabolism, for example, cardiovascular disease,stroke, diabetes, obesity, etc.

Further, dysfunction in the GPCRs described herein or in the levels ofthe ligands that bind to the GPCRs can produce a disease, condition, orsymptom associated with a lack of function of the particular cell typein which the GPCR is expressed. In certain embodiments, thepresently-described GPCRs can be used in the diagnosis and treatment ofcertain diseases or conditions, i.e., TGR-associated disorders. Forexample, the activity of GPCRs that are expressed or preferentiallyexpressed in a particular cell type (e.g., neurons), can be used tomodulate cellular function (e.g., responsiveness to extracellularsignals), thereby specifically modulating the function of the cells ofthat type in a patient. Further, mutations in the cell specific GPCRswill likely produce a disease, condition, or symptom associated with alack of function of the particular cell type.

Similarly, mutations or dysregulation of TGRs expressed in lymphocytesor hematopoietic cell-associated TGRs, i.e., TGRs preferentiallyexpressed in peripheral blood lymphocytes (PBLs), bone marrow, thymus,or hematopoietic cell lineages including cells involved in the immunesystem, can lead to malignancies, anemia, and other disorders of immunefunction such as autoimmune diseases (see, e.g., Harrison's Principlesof Internal Medicine, supra). Disorders in the function, or level, ofkidney-associated GPCRs will likely result in any of a number ofnephrotic conditions or diseases, such as renal failure, nephritis,nephrotic syndrome, asymptomatic urinary abnormalities, renal tubuledefects, hypertension, nephrolithiasis, or any other syndrome or diseaseassociated with the kidneys (see, e.g., Harrison's Principles ofInternal Medicine, 12th Edition, Wilson, et al., eds., McGraw-Hill,Inc.).

Mutation or dysregulation of adipocyte or liver GPCRs can lead todisorders relating to glucose metabolism, weight control andhyperlipidemia; and can also be used to detect, or diagnose a propensityfor, conditions such as obesity. Similarly, spleen-associated GPCRs maybe involved in any spleen-associated disorder or condition, e.g.,splenic enlargement, immune disorders, blood disorders, and others (see,e.g., Harrison's Principles of Internal Medicine, supra). Alteredfunction, or level, of GPCRs expressed in the colon can result in any ofa number of colon-associated conditions or diseases, e.g., inflammatorybowel disease such as Crohn's disease and ulcerative colitis, and otheralterations in bowel habit, rectal bleeding, pain, and other symptoms(see, e.g., Harrison's Principles of Internal Medicine, supra). Skeletalmuscle-associated GPCRs may play a role in various myopathies includingthose that cause muscle pain; acute, subacute, or chronic muscleweakness; and other diseases of the skeletal muscle system (see, e.g.,Harrison's Principles of Internal Medicine, supra). GPCRs expressed inthe heart may play a role heart failure, ischemic heart disease, andvarious cardiomyopathies (see, e.g., Harrison's Principles of InternalMedicine, supra).

GPCRs expressed in the brain or neural tissue can play a role in anynumber of disorders including neurological disease and neurodegenerativediseases as well as disorders in sensory perception, e.g., painperception (see, e.g., Harrison's Principles of Internal Medicine,supra). Disorders involving the brain include, but are not limited to,disorders involving neurons; disorders involving glia, such asastrocytes, oligodendrocytes, ependymal cells, and microglia;cerebrovascular diseases, such as those related to hypoxia, ischemia,and infarction; demyelinating diseases, such as multiple sclerosis;degenerative diseases, such as Alzheimer disease, Pick disease,Parkinson disease, and Huntington disease; and degenerative diseasesaffecting motor neurons, including amyotrophic lateral sclerosis; andvarious tumors involving neural tissue.

Mutation or altered activity of GPCRs preferentially expressed in thehypothalamus, will likely result in any number of conditions associatedwith the hypothalamus and the pituitary gland, which is often controlledby chemical mediators secreted by the hypothalamus. For example,dysfunction of hypothalamus-specific GPCRs can alter secretion of one ormore hypothalamic factors such as growth hormone-releasing hormone,somatostatin, gonadotropin-releasing hormone, thyrotropin-releasinghormone, and corticotropin-releasing hormone. Thus,hypothalamic-associated diseases include hypothyroidism, hypogonadism,growth disorders, and hyperprolactinemia, as well as diabetes insipidusand disturbances of thirst, sleep, temperature regulation, appetite,blood pressure or any other syndrome or disease associated with thehypothalamus (see, e.g., Harrison's Principles of Internal Medicine,12th Edition, Wilson, et al., eds., McGraw-Hill,

GPCRs that are expressed in reproductive tissues, e.g., ovaries, uterus,testis, may play a role in various disorders of the reproductive system(see, e.g., Harrison's Principles of Internal Medicine, supra). Theseinclude, for example, disorders involving the uterus and endometriumsuch as endometriosis; endometrial polyps; and various uterine tumor.Disorders of the ovary include amenorrhea, ovarian failure, and chronicanovulation. Disorders of the testis include infertility and disordersrelating to testosterone production.

GPCRs that are expressed in the adrenal glands may play a role indiseases related to adrenal function (see, e.g., Harrison's Principlesof Internal Medicine, supra). These include diseases of excess adrenalfunction, such as Cushing's syndrome, aldosteronism, and adrenalvirilism; as well as those disease of insufficient adrenal function,e.g., Addison's disease, secondary and acute adrenocorticalinsufficiency, and hypoaldosteronism.

GPCRs that are expressed predominantly in the kidney may play a role invarious diseases of the kidney (see, e.g., Harrison's Principles ofInternal Medicine, supra) or diseases related to kidney functionincluding hypertension, renal inflammatory disease, e.g.,glomerulonephritis, polycystic disease, and renal failure.

Accordingly, the methods of the invention can be used to diagnose any ofthe herein-described disorders or conditions in a patient, e.g., byexamining the sequence, level, or activity of any of the present GPCRsin a patient, wherein an alteration, e.g., a decrease, in the level ofexpression or activity in a GPCR, or the detection of a deleteriousmutation in a GPCR, indicates the presence or the likelihood of thedisease or condition. Further, modulation of the present GPCRs (e.g., byadministering modulators of the GPCR) can be used to treat or preventany of the conditions or diseases.

For example, TGR2 can be involved in inflammatory disorders anddisorders of the immune response. Furthermore, it can be involved innociceptive responses. Thus TGR2 modulators, e.g., β-alanine, β-alanineanalogs, other compounds that have the ability to compete with β-alaninefor binding to TGR2, or compounds that otherwise modulate β-alaninebinding or β-alanine-induced activity, may also be used for thetreatment of immune, inflammatory or nociceptive (pain) disorders. Paindisorders include, but are not limited, to neuropathic pain resultingfrom injury to specific nerves; pain associated with cancer, such aspain resulting from bone metastases in cancer; pain associated withinflammatory responses or inflammatory diseases, e.g., various types ofarthritis and other diseases including those listed below; and painassociated with chronic infections, such as ganglionic viral infections,e.g., Herpes infection. Acute and or/chronic pain due to injury, e.g.,burns, may also be treated. Immune and inflammatory disorders include avariety of disorders, e.g., chronic or acute inflammatory syndromes,such as rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis,gouty arthritis and other arthritic conditions; other autoimmunediseases, e.g., lupus erythematosus; pulmonary fibrosis; ileitis;colitis; Crohn's disease; pancreatitis, inflammatory responses or sepsisassociated with infection, e.g., viral or bacterial infection; dermalinflammatory responses, e.g., psoriasis, dermatoses, scleroderma,blistering disease; nephritis; neurogenic inflammation, e.g.,meningitis, septic shock, Down's syndrome, postischemic brain injury,HIV encephalopathy, Parkinson's disease, Alzheimer's disease,amyotrophic lateral sclerosis and multiple sclerosis; idiopathicinflammatory myopathies; inflammation of the blood vessels; reperfusioninjury; thyroiditis; Type I diabetes; allergies; graft vs. hostreaction, allograft rejections; and a variety of proliferative disordersof immune cells (see, e.g., Harrison's, supra) including, e.g., anemia,thrombocytopenia, leukopenia as well as immune malignancies such asleukemias and lymphomas.

GPR77 can be involved in disorders involving triglyceride metabolismand/or glucose metabolism. Additionally, it may also be involved indisorders of the brain, e.g., disorders of the hypothalamus or cortex.Thus, modulators of GPR77, e.g., compounds that have the ability tocompete with ASP for binding to GPR77, or compounds that otherwisemodulate ASP binding or ASP-induced activity, may be used to treat suchdisorders. These disorders include, but are not limited to, disorders offat metabolism, obesity, diabetes, atherosclerosis, and other diseasesrelated to triglyceride metabolism. Other disorders, such asneurological disorders and hormonal disorders, e.g., those stemming fromhypothalamic dysfunction, may also be treated with compounds thatmodulate ASP binding to GPR77.

LGR4, LGR5, and LGR6 can be involved in disorders relating to calciumuptake, growth, reproduction, wound healing, atherogenesis,angiogenesis, neuronal differentiation, and various neurologicaldisorders. Accordingly, modulators of these receptors, e.g.,stanniocalcins or other compounds that have the ability to compete witha stanniocalcin for binding to the GPCR, or compounds that otherwisemodulate stanniocalcin binding to its receptor or stanniocalcin-inducedactivity may be used to treat such conditions. These conditions include,but are not limited to disorders of bone growth and regeneration, e.g.,osteoporosis, bone fractures, bone loss associated with periodontitis;kidney disorders; disorders of mineral metabolism; atherogenesis;angiogenesis; wound healing, conditions or diseases that relate togrowth, such as delayed or excessive growth; conditions or diseases thatrelate to reproduction, such as infertility; neurological disordersincluding ischemic brain injury; and other neurological diseases such asthose that involve inflammation.

Modulators of GPR40 and GPR43 may also be used for the treatment ofconditions relating to fat metabolism, for example, dyslipidemia,coronary artery disease, atherosclerosis, obesity, thrombosis, angina,chronic renal failure, peripheral vascular disease, stroke, type IIdiabetes and metabolic syndrome (syndrome X). Further, GPR40 isexpressed in the brain, in particular, in the substantia nigra andspinal cord. Thus, modulators of GPR40 that are identified as disclosedherein may be used for the treatment of other neurological diseases, aswell as diseases such as ischemic brain injury, stroke.

Modulators of TGR18 and TGR164 may be used for the treatment ofconditions relating to kidney dysfunction including including Bartter'ssyndrome, Gitelman syndrome, nephrolithiasis, renal amyloidosis,hypertension; primary aldosteronism; Addison's disease; renal failure;glomerulonephritis; chronic glomerulonephritis: tubulointerstitialnephritis; cystic disorders of the kidney and dysplastic malformationssuch as polycystic disease, renal dysplasias, and cortical or medullarycysts; inherited polycystic renal diseases (PRD), such as recessive andautosomal dominant PRD; medullary cystic disease; medullary spongekidney and tubular dysplasia; Alport's syndrome; non-renal cancers whichaffect renal physiology, such as bronchogenic tumors of the lungs ortumors of the basal region of the brain; multiple myeloma;adenocarcinomas of the kidney; metastatic renal carcinoma; in addition,nephrotoxic disorders include any functional or morphologic change inthe kidney produced by any pharmaceutical, chemical, or biological agentthat is ingested, injected, inhaled, or absorbed. Some broad categoriesof common nephrotoxic agents are heavy metals, all classes ofantibiotics, analgesics, solvents, oxalosis-inducing agents, anticancerdrugs, herbicides and pesticides, botanicals and biologicals, andantiepileptics.

Administration and Pharmaceutical Compositions

Modulators of the GPCR-ligand interaction can be administered to amammalian subject for modulation of signal transduction in vivo, e.g.,for the treatment of any of the diseases or conditions described supra.As described in detail below, the modulators are administered in anysuitable manner, optionally with pharmaceutically acceptable carriers.

The identified modulators can be administered to a patient attherapeutically effective doses to prevent, treat, or control diseasesand disorders mediated, in whole or in part, by a GPCR-ligandinteraction of the present invention. The compositions are administeredto a patient in an amount sufficient to elicit an effective protectiveor therapeutic response in the patient. An amount adequate to accomplishthis is defined as “therapeutically effective dose.” The dose will bedetermined by the efficacy of the particular GPCR modulators employedand the condition of the subject, as well as the body weight or surfacearea of the area to be treated. The size of the dose also will bedetermined by the existence, nature, and extent of any adverse effectsthat accompany the administration of a particular compound or vector ina particular subject.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, for example, by determining the LD₅₀ (the dose lethal to 50% ofthe population) and the ED₅₀ (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index and can be expressed as the ratio, LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue to minimize potential damage to normal cellsand, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can beused to formulate a dosage range for use in humans. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage can varywithin this range depending upon the dosage form employed and the routeof administration. For any compound used in the methods of theinvention, the therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose can be formulated in animal models toachieve a circulating plasma concentration range that includes the IC₅₀(the concentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma can be measured, for example, by high performance liquidchromatography (HPLC). In general, the dose equivalent of a modulator isfrom about 1 ng/kg to 10 mg/kg for a typical subject.

Pharmaceutical compositions for use in the present invention can beformulated by standard techniques using one or more physiologicallyacceptable carriers or excipients. The compounds and theirphysiologically acceptable salts and solvates can be formulated foradministration by any suitable route, including via inhalation,topically, nasally, orally, parenterally (e.g., intravenously,intraperitoneally, intravesically or intrathecally) or rectally.

For oral administration, the pharmaceutical compositions can take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients, including binding agents,for example, pregelatinised maize starch, polyvinylpyrrolidone, orhydroxypropyl methylcellulose; fillers, for example, lactose,microcrystalline cellulose, or calcium hydrogen phosphate; lubricants,for example, magnesium stearate, talc, or silica; disintegrants, forexample, potato starch or sodium starch glycolate; or wetting agents,for example, sodium lauryl sulphate. Tablets can be coated by methodswell known in the art. Liquid preparations for oral administration cantake the form of, for example, solutions, syrups, or suspensions, orthey can be presented as a dry product for constitution with water orother suitable vehicle before use. Such liquid preparations can beprepared by conventional means with pharmaceutically acceptableadditives, for example, suspending agents, for example, sorbitol syrup,cellulose derivatives, or hydrogenated edible fats; emulsifying agents,for example, lecithin or acacia; non-aqueous vehicles, for example,almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils;and preservatives, for example, methyl or propyl-p-hydroxybenzoates orsorbic acid. The preparations can also contain buffer salts, flavoring,coloring, and/or sweetening agents as appropriate. If desired,preparations for oral administration can be suitably formulated to givecontrolled release of the active compound.

For administration by inhalation, the compounds may be convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, forexample, dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In thecase of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, for example, gelatin for use in an inhaler or insufflator can beformulated containing a powder mix of the compound and a suitable powderbase, for example, lactose or starch.

The compounds can be formulated for parenteral administration byinjection, for example, by bolus injection or continuous infusion.Formulations for injection can be presented in unit dosage form, forexample, in ampoules or in multi-dose containers, with an addedpreservative. The compositions can take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and can containformulatory agents, for example, suspending, stabilizing, and/ordispersing agents. Alternatively, the active ingredient can be in powderform for constitution with a suitable vehicle, for example, sterilepyrogen-free water, before use.

The compounds can also be formulated in rectal compositions, forexample, suppositories or retention enemas, for example, containingconventional suppository bases, for example, cocoa butter or otherglycerides.

Furthermore, the compounds can be formulated as a depot preparation.Such long-acting formulations can be administered by implantation (forexample, subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, the compounds can be formulated withsuitable polymeric or hydrophobic materials (for example as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

The compositions can, if desired, be presented in a pack or dispenserdevice that can contain one or more unit dosage forms containing theactive ingredient. The pack can, for example, comprise metal or plasticfoil, for example, a blister pack. The pack or dispenser device can beaccompanied by instructions for administration.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

Example 1 Identification of β-Alanine as a Ligand for TGR2

A library of natural ligands was screened for the ability to activateTGR2. β-alanine was a positive hit at 10 μM in an Aequorin assay (see,e.g., An, et al., Proc. Natl. Acad. Sci USA 98:7576, 2001) that measuresTGR2-mediated increase in intracellular calcium concentration. Additionof β-alanine at 10 μM did not activate 40 other orphan GPCRs that weretested, which suggested that β-alanine is specific for TGR2. β-alaninealso activated TGR2 in an inositol phosphate accumulation assay, andalso specifically activated the mouse ortholog mMrgD in an Aequorinassay. Dose-dependence analysis gave an EC₅₀ of 9.1 μM for β-alanine inan Aequorin assay using CHO cells transiently transfected with TGR2(FIG. 1).

Tissue-specific expression using quantitative PCR demonstrated that TGR2is expressed in human immune cells and tissues, e.g., T lymphocytes andthymus.

Example 2 Identification of ASP as a Natural Ligand for GPR77

GPR77 has been shown to bind to complement factors C5a and C3a. Using acompetitive binding assay, ASP was also shown to bind to GPR77.

GPR77 was transiently transfected into cells. A competition assay usinga membrane binding format showed that ASP competed with C5a and C3a forbinding to GPR77.

Example 3 Identification of Stanniocalcins as Natural Ligands for LGR4,LGR5, and LGR6

Stanniocalcins were identified as natural ligands for LGR4, LGR5, andLGR6.

Example 4 Identification of Natural Ligands for GPR43

A battery of fatty acids were tested for the ability to activate GPR43.Short chain fatty acids of 2-3 carbons in length activated GPR43. Inparticular, GPR43 is activated by acetate and propionate at about 140 μM(FIG. 2). The medium chain fatty acids pentanoate, hexanoate,heptanoeate, octanoate, and nonanoate do not activate GPR43 in thisexperiment.

Example 5 Identification of Natural Ligands for GPR40

Fatty acids and other molecules were also tested for the ability toactivate GPR40. The results showed that medium and long chain (6C's orlonger) saturated fatty acids activate GPR40 (FIG. 3). Furthermore,polyunsaturated fatty acids activate GPR40 (FIG. 4). Additional analysesdemonstrated that hypolipidemic drugs and fibrates, a class oflipid-lowering agents also activate GPR40 (FIG. 4).

Example 6 Identification of Natural Ligands of TGR18

Succinic acid was identified as a natural ligand of TGR18. The ligandwas purified from kidney as follows. During purification, activity wasmonitored using an Aequorin assay of CHO cells transiently transfectedwith TGR18.

Purification of TGR18 Ligand

Porcine kidney (6 kg) tissue was homogenized and extracted inethanol/water/acetic acid, 50/46/4, v/v/v. After centrifugation, thesupernatant was filtered and lyophilized to remove ethanol and aceticacid. The lyophilized material was resuspended in 10 mM K₂HPO₄ pH8.0,loaded onto an XK50/20 Q sepharose anion-exchange column, and elutedwith buffer A (10 mM K₂HPO₄ pH 8.0) followed by a linear gradient ofbuffer A to buffer B (50 mM K₂HPO₄ pH 8.0, 0.25M NaCl). Active fractionswere pooled and fractionated on a Hiload 26/60 Superdex 30size-exclusion column in Hank's buffered saline (HBSS). The activefractions were pooled, and loaded onto a Superdex peptide HR 10/30size-exclusion column pre-equilibrated with 0.1% TFA/H₂O and elutedisocratically with the same buffer. The active fractions were pooled,concentrated, injected on a ODS-AQ 4.6×250 mm column, and elutedisocratically with 0.1% TFA/H₂O. The active fractions were lyophilizedand dissolved in D₂O for NMR and Mass spectrometry analysis.

NMR and Mass Spectrometry

The final purified TGR18 ligand preparation was dissolved in D₂O and the¹H/¹³C NMR spectra were recorded on a Bruker DRX700 spectrometer. Asingle ¹H signal was observed at 2.66 ppm. Two signals were observed at31.8 ppm and 179.9 ppm in the decoupled ¹³C spectrum. In the coupled ¹³Cspectrum, the signal at 31.8 ppm shows a triplet of triplets splittingpattern, implying two adjacent CH₂ groups with magnetic equivalence. Thecarboxyl groups were suggested to attach to the CH₂ groups based on thetypical chemical shift at 179.9 ppm. The final structure was confirmedby comparison of NMR data from the TGR18 ligand preparation and succinicacid.

Positive ion electrospray ionization mass spectrometry was applied toanalyze the final purified TGR18 ligand preparation. The mass spectrumwas obtained on a HP 1100MSD spectrometer which was operated under unitmass resolution conditions across the mass range of interest. Full-scanmass spectrum covering m/z of 50-500 was acquired. The observation ofthe molecular ion ([M+1]+, 119.2) confirms succinic acid as the TGR18ligand.

Aequorin Assay

In order to examine the ability of a compound to activate TGR18, CHOcells were transiently transfected with 10 μg of the GPCR and 10 μgAequorin reporter gene in a 150 mm dish. As a negative control, the sameamount of empty vector and the Aequorin reporter were cotransfected intoCHO cells. After 24 hours, cells were harvested and resuspended inAequorin buffer (Hank's buffered saline with 20 mM Hepes, pH7.6 and 0.1%BSA) containing 1 μg/ml coelenterazine f and incubated at roomtemperature for a further 2 hours. The Aequorin luminescence wasrecorded on Microlumat after injecting 100 μl of cells into 100 μl ofligand prepared in Aequorin buffer. The results showed that succinicacid and related compounds activated TGR18. Human, mouse, and rat TGR18were all activated by succinic acid (FIG. 6A). In a specificity test,succinic acid at 100 μM does not activate thirty other GPCRs tested.

Fluorometric Imaging Plate Reader (FLIPR) Assay

Mobilization of intracellular calcium in response to various ligands wasalso evaluated using a FLIPR assay. In this assay, 293 cells stablyexpressing TGR18 were seeded into 384-well plates and incubated at 37°C. overnight. The growth medium was then aspirated and replaced with 50μl loading medium (FLIPR no-wash kit, Molecular Devices) and incubatedat 37° C. for 1 hour. The cells were placed in a fluorometric imagingplate reader (FLIPR), and changes in cellular fluorescence were recordedafter the addition of 25 μl of various ligands diluted in FLIPR buffer(FLIPR no-wash kit, Molecular Devices). The results showed that succinicacid activated TGR18 at an EC₅₀ of about 27 μM in the experimentpresented in FIG. 6B. Analogs of succinic acid that activated TGR18include succinic acid, maleic acid, oxalacetic acid, methylmalonic acidand itaconic acid (FIG. 6C).

Inositol Phosphate Accumulation Assay

The ability of various ligands to increase inositol phosphate was alsoassessed. For this analysis, 293 cells stably expression TGR18 wereincubated in inositol-free DMEM/10% dialyzed FCS/1 μCi/ml ³H-inositolfor 16 hours. Following incubation 10 mM LiCl was added to the cells for15 minutes. Cells were then stimulated with various ligands for 45minutes and extracted with ice-cold 20 mM formic acid. H3-inositolphosphate was collected on Dowex ion-exchange column (formate form) andradioactivity recorded by Topcount scintillation counter. The resultsshowed that succinic acid increased inositol phosphate levels, whereasother dicarboxylic acids, e.g., fumaric acid, did not.

Luciferase Reporter Assay

The ability of a ligand to induce TGR18-mediated activation of aCRE-luciferase reporter was also evaluated. For this assay, 293 cellswere transiently transfected with CRE-luciferase reporter, tk-renilaluciferase reporter and TGR18 plasmid. Succinic acid was then added tothe cell culture supernatants and incubated for a further 6 hours. Theluminescence were recorded on CLIPR after cell lysis as illustrated inDual-luciferase assay kit (Promega). The results show that succinic acidinduced TGR18-induced luciferase reporter activity.

Example 7 Identification of the Natural Ligand for TGR164

A screening of about 50 carboxylic acids using Aequorin assays, whichwere performed using the methodology described in Example 6, identifiedα-ketoglutaric acid as a TGR164 ligand Aequorin assays also showed thatitaconic acid (FIGS. 7A and 7B) activated TGR164. Inositol phosphateaccumulation, performed as described in Example 6, was also observed inresponse to α-ketoglutaric acid and itaconic acid-induced activation ofTGR164.

Analysis of TGR164 activation in multiple cell lines using Aequorinassays as described in Example 6 showed that α-ketoglutaric acidactivates TGR164 in various cell lines.

All publications, accession numbers, patents, and patent applicationscited in this specification are herein incorporated by reference as ifeach individual publication or patent application were specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims. TABLE OF GPCR NUCLEIC ACID AND PROTEINSEQUENCES SEQ ID NO:1 Human TGR2 Nucleic AcidATGAACCAGACTTTGAATAGCAGTGGGACCGTGGAGTCAGCCCTAAACTATTCCAGAGGGAGCACAGTGCACACGGCCTACCTGGTGCTGAGCTCCCTGGCCATGTTCACCTGCCTGTGCGGGATGGCAGGCAACAGCATGGTGATCTGGCTGCTGGGCTTTCGAATGCACAGGAACCCCTTCTGCATCTATATCCTCAACCTGGCGGCAGCCGACCTCCTCTTCCTCTTCAGCATGGCTTCCACGCTCAGCCTGGAAACCCAGCCCCTGGTCAATACCACTGACAAGGTCCACGAGCTGATGAAGAGACTGATGTACTTTGCCTACACAGTGGGCCTGAGCCTGCTGACGGCCATCAGCACCCAGCGCTGTCTCTCTGTCCTCTTCCCTATCTGGTTCAAGTGTCACCGGCCCAGGCACCTGTCAGCCTGGGTGTGTGGCCTGCTGTGGACACTCTGTCTCCTGATGAACGGGTTGACCTCTTCCTTCTGCAGCAAGTTCTTGAAATTCAATGAAGATCGGTGCTTCAGGGTGGACATGGTCCAGGCCGCCCTCATCATGGGGGTCTTAACCCCAGTGATGACTCTGTCCAGCCTGACCCTCTTTGTCTGGGTGCGGAGGAGCTCCCAGCAGTGGCGGCGGCAGCCCACACGGCTGTTCGTGGTGGTCCTGGCCTCTGTCCTGGTGTTCCTCATCTGTTCCCTGCCTCTGAGCATCTACTGGTTTGTGCTCTACTGGTTGAGCCTGCCGCCCGAGATGCAGGTCCTGTGCTTCAGCTTGTCACGCCTCTCCTCGTCCGTAAGCAGCAGCGCCAACCCCGTCATCTACTTCCTGGTGGGCAGCCGGAGGAGCCACAGGCTGCCCACCAGGTCCCTGGGGACTGTGCTCCAACAGGCGCTTCGCGAGGAGCCCGAGCTGGAAGGTGGGGAGACGCCCACCGTGGGCACCAATGAGATGGGGGCT SEQ ID NO:2 HumanTGR2 Protein sequenceMNQTLNSSGTVESALNYSRGSTVHTAYLVLSSLAMFTCLCGMAGNSMVIWLLGFRMHRNPFCIYILNLAAADLLFLFSMASTLSLETQPLVNTTDKVHELMKRLMYFAYTVGLSLLTAISTQRCLSVLFPIWFKCHRPRHLSAWVCGLLWTLCLLMNGLTSSFCSKFLKFNEDRCFRVDMVQAALIMGVLTPVMTLSSLTLFVWVRRSSQQWRRQPTRLFVVVLASVLVFLICSLPLSIYWFVLYWLSLPPEMQVLCFSLSRLSSSVSSSANPVIYFLVGSRRSHRLPTRSLGTVLQQALREEPELEGGETPTVGTNEMGA SEQ IDNO:3 Human GPR77 nucleic acid sequence   1atggggaacgattctgtcagctacgagtatggggattacagcgacctctcggaccgccct  61gtggactgcctggatggcgcctgcctggccatcgacccgctgcgcgtggccccgctccca 121ctgtatgccgccatcttcctggtgggggtgccgggcaatgccatggtggcctgggtggct 181gggaaggtggcccgccggagggtgggtgccacctggttgctccacctggccgtggcggat 241ttgctgtgctgtttgtctctgcccatcctggcagtgcccattgcccgtggaggccactgg 301ccgtatggtgcagtgggctgtcgggcgctgccctccatcatcctgctgaccatgtatgcc 361agcgtcctgctcctggcagctctcagtgccgacctctgcttcctggctctcgggcctgcc 421tggtggtctacggttcagcgggcgtgcggggtgcaggtggcctgtggggcagcctggaca 481ctggccttgctgctcaccgtgccctccgccatctaccgccggctgcaccaggagcacttc 541ccagcccggctgcagtgtgtggtggactacggcggctcctccagcaccgagaatgcggtg 601actgccatccggtttctttttggcttcctggggcccctggtggccgtggccagctgccac 661agtgccctcctgtgctgggcagcccgacgctgccggccgctgggcacagccattgtggtg 721gggttttttgtctgctgggcaccctaccacctgctggggctggtgctcactgtggcggcc 781ccgaactccgcactcctggccagggccctgcgggctgaacccctcatcgtgggccttgcc 841ctcgctcacagctgcctcaatcccatgctcttcctgtattttgggagggctcaactccgc 901cggtcactgccagctgcctgtcactgggccctgagggagtcccagggccaggacgaaagt 961gtggacagcaagaaatccaccagccatgacctggtctcggagatggaggtgtag SEQ ID NO:4 HumanGPR77protein sequenceMGNDSVSYEYGDYSDLSDRPVDCLDGACLAIDPLRVAPLPLYAAIFLVGVPGNAMVAWVAGKVARRRVGATWLLHLAVADLLCCLSLPILAVPIARGGHWPYGAVGCRALPSIILLTMYASVLLLAALSADLCFLALGPAWWSTVQRACGVQVACGAAWTLALLLTVPSAIYRRLHQEHFPARLQCVVDYGGSSSTENAVTAIRFLFGFLGPLVAVASCHSALLCWAARRCRPLGTAIVVGFFVCWAPYHLLGLVLTVAAPNSALLARALRAEPLIVGLALAHSCLNPMLFLYFGRAQLRRSLPAACHWALRESQGQDESVDSKKSTSHDLVSEMEV SEQ ID NO:5 Human LGR4 nucleic acid sequence atggtgcagcagttcc ccaatcttac aggaactgtc cacctggaaa gtctgacttt gacaggtacaaagataagca gcatacctaa taatttgtgt caagaacaaa agatgcttag gactttggacttgtcttaca ataatataag agaccttcca agttttaatg gttgccatgc tctggaagaaatttctttac agcgtaatca aatctaccaa ataaaggaag gcacctttca aggcctgatatctctaagga ttctagatct gagtagaaac ctgatacatg aaattcacag tagagcttttgccacacttg ggccaataac taacctagat gtaagtttca atgaattaac ttcctttcctacggaaggcc tgaatgggct aaatcaactg aaacttgtgg gcaacttcaa gctgaaagaagccttagcag caaaagactt tgttaacctc aggtctttat cagtaccata tgcttatcagtgctgtgcat tttggggttg tgactcttat gcaaatttaa acacagaaga taacagcctccaggaccaca gtgtggcaca ggagaaaggt actgctgatg cagcaaatgt cacaagcactcttgaaaatg aagaacatag tcaaataatt atccattgta caccttcaac aggtgcttttaagccctgtg aatatttact gggaagctgg atgattcgtc ttactgtgtg gttcattttcttggttgcat tatttttcaa cctgcttgtt attttaacaa catttgcatc ttgtacatcactgccttcgt ccaaattgtt tataggcttg atttctgtgt ctaacttatt catgggaatctatactggca tcctaacttt tcttgatgct gtgtcctggg gcagattcgc tgaatttggcatttggtggg aaactggcag tggctgcaaa gtagctgggt ttcttgcagt tttctcctcagaaagtgcca tatttttatt aatgctagca actgtcgaaa gaagcttatc tgcaaaagatataatgaaaa atgggaagag caatcatctc aaacagttcc gggttgctgc ccttttggctttcctaggtg ctacagtagc aggctgtttt ccccttttcc atagagggga atattctgcatcaccccttt gtttgccatt tcctacaggt gaaacgccat cattaggatt cactgtaacgttagtgctat taaactcact agcattttta ttaatggccg ttatctacac taaactatactgcaacttgg aaaaagagga cctctcagaa aactcacaat ctagcatgat taagcatgtcgcttggctaa tcttcaccaa ttgcatcttt ttctgccctg tggcgttttt ttcatttgcaccattgatca ctgcaatctc tatcagcccc gaaataatga agtctgttac tctgatattttttccattgc ctgcttgcct gaatccagtc ctgtatgttt tcttcaaccc aaagtttaaagaagactgga agttactgaa gcgacgtgtt accaagaaaa gtggatcagt ttcagtttccatcagtagcc aaggtggttg tctggaacag gatttctact acgactgtgg catgtactcacatttgcagg gcaacctgac tgtttgcgac tgctgcgaat cgtttctttt aacaaagccagtatcatgca aacacttgat aaaatcacac agctgtcctg cattggcagt ggcttcttgccaaagacctg agggctactg gtccgactgt ggcacacagt cggcccactc tgattatgcagatgaagaag attcctttgt ctcagacagt tctgaccagg tgcaggcctg tggacgagcctgcttctacc agagtagagg attccctttg gtgcgctatg cttacaatct accaagagttaaagactga SEQ ID NO:6 Human LGR4 protein sequenceMVQQFPNLTGTVHLESLTLTGTKISSIPNNLCQEQKMLRTLDLSYNNIRDLPSFNGCHALEEISLQRNQIYQIKEGTFQGLISLRILDLSRNLIHEIHSRAFATLGPITNLDVSFNELTSFPTEGLNGLNQLKLVGNFKLKEALAAKDFVNLRSLSVPYAYQCCAFWGCDSYANLNTEDNSLQDHSVAQEKGTADAANVTSTLENEEHSQIIIHCTPSTGAFKPCEYLLGSWMIRLTVWFIFLVALFFNLLVILTTFASCTSLPSSKLFIGLISVSNLFMGIYTGILTFLDAVSWGRFAEFGIWWETGSGCKVAGFLAVFSSESAIFLLMLATVERSLSAKDIMKNGKSNHLKQFRVAALLAFLGATVAGCFPLFHRGEYSASPLCLPFPTGETPSLGFTVTLVLLNSLAFLLMAVIYTKLYCNLEKEDLSENSQSSMIKHVAWLIFTNCIFFCPVAFFSFAPLITAISISPEIMKSVTLIFFPLPACLNPVLYVFFNPKFKEDWKLLKRRVTKKSGSVSVSISSQGGCLEQDFYYDCGMYSHLQGNLTVCDCCESFLLTKPVSCKHLIKSHSCPALAVASCQRPEGYWSDCGTQSAHSDYADEEDSFVSDSSDQVQACGRACFYQSRGFPLVRYAYNLPRVKD SEQ ID NO:7Human LGR5 nucleic acid sequence    1atggacacctcccggctcggtgtgctcctgtccttgcctgtgctgctgcagctggcgacc   61gggggcagctctcccaggtctggtgtgttgctgaggggctgccccacacactgtcattgc  121gagcccgacggcaggatgttgctcagggtggactgctccgacctggggctctcggagctg  181ccttccaacctcagcgtcttcacctcctacctagacctcagtatgaacaacatcagtcag  241ctgctcccgaatcccctgcccagtctccgcttcctggaggagttacgtcttgcgggaaac  301gctctgacatacattcccaagggagcattcactggcctttacagtcttaaagttcttatg  361ctgcagaataatcagctaagacacgtacccacagaagctctgcagaatttgcgaagcctt  421caatccctgcgtgtggatgctaaccacatcagctatgtgcccccaagctgtttcagtggc  481ctgcattccctgaggcacctgtggctggatgacaatgcgttaacagaaatccccgtccag  541gcttttagaagtttatcggcattgcaagccatgaccttggccctgaacaaaatacaccac  601ataccagactatgcctttggaaacctctccagcttggtagttctacatctccataacaat  661agaatccactccctgggaaagaaatgctttgatgggctccacagcctagagactttagat  721ttaaattacaataaccttgatgaattccccactgcaattaggacactctccaaccttaaa  781gaactaggatttcatagcaacaatatcaggtcgatacctgagaaagcatttgtaggcaac  841ccttctcttattacaatacatttctatgacaatcccatccaatttgttgggagatctgct  901tttcaacatttacctgaactaagaacactgactctgaatggtgcctcacaaataactgaa  961tttcctgatttaactggaactgcaaacctggagagtctgactttaactggagcacagatc 1021tcatctcttcctcaaaccgtctgcaatcagttacctaatctccaagtgctagatctgtct 1081tacaacctattagaagatttacccagtttttcagtctgccaaaagcttcagaaaattgac 1141ctaagacataatgaaatctacgaaattaaagttgacactttccagcagttgcttagcctc 1201cgatcgctgaatttggcttggaacaaaattgctattattcaccccaatgcattttccact 1261ttgccatccctaataaagctggacctatcgtccaacctcctgtcgtcttttcctataact 1321gggttacatggtttaactcacttaaaattaacaggaaatcatgccttacagagcttgata 1381tcatctgaaaactttccagaactcaaggttatagaaatgccttatgcttaccagtgctgt 1441gcatttggagtgtgtgagaatgcctataagatttctaatcaatggaataaaggtgacaac 1501agcagtatggacgaccttcataagaaagatgctggaatgtttcaggctcaagatgaacgt 1561gaccttgaagatttcctgcttgactttgaggaagacctgaaagcccttcattcagtgcag 1621tgttcaccttccccaggccccttcaaaccctgtgaacacctgcttgatggctggctgatc 1681agaattggagtgtggaccatagcagttctggcacttacttgtaatgctttggtgacttca 1741acagttttcagatcccctctgtacatttcccccattaaactgttaattggggtcatcgca 1801gcagtgaacatgctcacgggagtctccagtgccgtgctggctggtgtggatgcgttcact 1861tttggcagctttgcacgacatggtgcctggtgggagaatggggttggttgccatgtcatt 1921ggttttttgtccatttttgcttcagaatcatctgttttcctgcttactctggcagccctg 1981gagcgtgggttctctgtgaaatattctgcaaaatttgaaacgaaagctccattttctagc 2041ctgaaagtaatcattttgctctgtgccctgctggccttgaccatggccgcagttcccctg 2101ctgggtggcagcaagtatggcgcctcccctctctgcctgcctttgccttttggggagccc 2161agcaccatgggctacatggtcgctctcatcttgctcaattccctttgcttcctcatgatg 2221accattgcctacaccaagctctactgcaatttggacaagggagacctggagaatatttgg 2281gactgctctatggtaaaacacattgccctgttgctcttcaccaactgcatcctaaactgc 2341cctgtggctttcttgtccttctcctctttaataaaccttacatttatcagtcctgaagta 2401attaagtttatccttctggtggtagtcccacttcctgcatgtctcaatccccttctctac 2461atcttgttcaatcctcactttaaggaggatctggtgagcctgagaaagcaaacctacgtc 2521tggacaagatcaaaacacccaagcttgatgtcaattaactctgatgatgtcgaaaaacag 2581tcctgtgactcaactcaagccttggtaacctttaccagctccagcatcacttatgacctg 2641cctcccagttccgtgccatcaccagcttatccagtgactgagagctgccatctttcctct 2701gtggcatttgtcccatgtctctaa SEQ ID NO:8 Human LGR5 protein sequenceMDTSRLGVLLSLPVLLQLATGGSSPRSGVLLRGCPTHCHCEPDGRMLLRVDCSDLGLSELPSNLSVFTSYLDLSMNNISQLLPNPLPSLRFLEELRLAGNALTYIPKGAFTGLYSLKVLMLQNNQLRHVPTEALQNLRSLQSLRLDANHISYVPPSCFSGLHSLRHLWLDDNALTEIPVQAFRSLSALQAMTLALNKIHHIPDYAFGNLSSLVVLHLHNNRIHSLGKKCFDGLHSLETLDLNYNNLDEFPTAIRTLSNLKELGFHSNNIRSIPEKAFVGNPSLITIHFYDNPIQFVGRSAFQHLPELRTLTLNGASQITEFPDLTGTANLESLTLTGAQISSLPQTVCNQLPNLQVLDLSYNLLEDLPSFSVCQKLQKIDLRHNEIYEIKVDTFQQLLSLRSLNLAWNKIAIIHPNAFSTLPSLIKLDLSSNLLSSFPITGLHGLTHLKLTGNHALQSLISSENFPELKVIEMPYAYQCCAFGVCENAYKISNQWNKGDNSSMDDLHKKDAGMFQAQDERDLEDFLLDFEEDLKALHSVQCSPSPGPFKPCEHLLDGWLIRIGVWTIAVLALTCNALVTSTVFRSPLYISPIKLLIGVIAAVNMLTGVSSAVLAGVDAFTFGSFARHGAWWENGVGCHVIGFLSIFASESSVFLLTLAALERGFSVKYSAKFETKAPFSSLKVIILLCALLALTMAAVPLLGGSKYGASPLCLPLPFGEPSTMGYMVALILLNSLCFLMMTIAYTKLYCNLDKGDLENIWDCSMVKHIALLLFTNCILNCPVAFLSFSSLINLTFISPEVIKFILLVVVPLPACLNPLLYILFNPHFKEDLVSLRKQTYVWTRSKHPSLMSINSDDVEKQSCDSTQALVTFTSSSITYDLPPSSVPSPAYPVTESCHLSSVAFVPCL SEQ IDNO:9 Human LGR6 nucleic acid sequence    1atgcgcttggagggagagggccgctcagcgagggcgggacagaatctctcccgggctggg   61agtgcacggcgcggtgcgcccagggacctcagcatgaacaacctcacagagcttcagcct  121ggcctcttccaccacctgcgcttcttggaggagctgcgtctctctgggaaccatctctca  181cacatcccaggacaagcattctctggtctctacagcctgaaaatcctgatgctgcagaac  241aatcagctgggaggaatccccgcagaggcgctgtgggagctgccgagcctgcagtcgcta  301gacctgaattataacaagctgcaggagttccctgtggccatccggaccctgggcagactg  361caggaactggggttccataacaacaacatcaaggccatcccagaaaaggccttcatgggg  421aaccctctgctacagacgatacacttttatgataacccaatccagtttgtgggaagatcg  481gcattccagtacctgcctaaactccacacactatctctgaatggtgccatggacatccag  541gagtttccagatctcaaaggcaccaccagcctggagatcctgaccctgacccgcgcaggc  601atccggctgctcccatcggggatgtgccaacagctgcccaggctccgagtcctggaactg  661tctcacaatcaaattgaggagctgcccagcctgcacaggtgtcagaaattggaggaaatc  721ggcctccaacacaaccgcatctgggaaattggagctgacaccttcagccagctgagctcc  781ctgcaagccctggatcttagctggaacgccatccggtccatccaccccgaggccttctcc  841accctgcactccctggtcaagctggacctgacagacaaccagctgaccacactgcccctg  901gctggacttgggggcttgatgcatctgaagctcaaagggaaccttgctctctcccaggcc  961ttctccaaggacagtttcccaaaactgaggatcctggaggtgccttatgcctaccagtgc 1021tgtccctatgggatgtgtgccagcttcttcaaggcctctgggcagtgggaggctgaagac 1081cttcaccttgatgatgaggagtcttcaaaaaggcccctgggcctccttgccagacaagca 1141gagaaccactatgaccaggacctggatgagctccagctggagatggaggactcaaagcca 1201caccccagtgtccagtgtagccctactccaggccccttcaagccctgtgagtacctcttt 1261gaaagctggggcatccgcctggccgtgtgggccatcgtgttgctctccgtgctctgcaat 1321ggactggtgctgctgaccgtgttcgctggcgggcctgtccccctgcccccggtcaagttt 1381gtggtaggtgcgattgcaggcgccaacaccttgactggcatttcctgtggccttctagcc 1441tcagtcgatgccctgacctttggtcagttctctgagtacggagcccgctgggagacgggg 1501ctaggctgccgggccactggcttcctggcagtacttgggtcggaggcatcggtgctgctg 1561ctcactctggccgcagtgcagtgcagcgtctccgtctcctgtgtccgggcctatgggaag 1621tccccctccctgggcagcgttcgagcaggggtcctaggctgcctggcactggcagggctg 1681gccgccgcactgcccctggcctcagtgggagaatacggggcctccccactctgcctgccc 1741tacgcgccacctgagggtcagccagcagccctgggcttcaccgtggccctggtgatgatg 1801aactccttctgtttcctggtcgtggccggtgcctacatcaaactgtactgtgacctgccg 1861cggggcgactttgaggccgtgtgggactgcgccatggtgaggcacgtggcctggctcatc 1921ttcgcagacgggctcctctactgtcccgtggccttcctcagcttcgcctccatgctgggc 1981ctcttccctgtcacgcccgaggccgtcaagtctgtcctgctggtggtgctgcccctgcct 2041gcctgcctcaacccactgctgtacctgctcttcaacccccacttccgggatgaccttcgg 2101cggcttcggccccgcgcaggggactcagggcccctagcctatgctgcggccggggagctg 2161gagaagagctcctgtgattctacccaggccctggtagccttctctgatgtggatctcatt 2221ctggaagcttctgaagctgggcggccccctgggctggagacctatggcttcccctcagtg 2281accctcatctcctgtcagcagccaggggcccccaggctggagggcagccattgtgtagag 2341ccagaggggaaccactttgggaacccccaaccctccatggatggagaactgctgctgagg 2401gcagagggatctacgccagcaggtggaggcttgtcagggggtggcggctttcagccctct 2461ggcttggccttgcttcacacgtat SEQ ID NO:10 Human LGR6 protein sequenceMRLEGEGRSARAGQNLSRAGSARRGAPRDLSMNNLTELQPGLFHHLRFLEELRLSGNHLSHIPGQAFSGLYSLKILMLQNNQLGGIPAEALWELPSLQSLDLNYNKLQEFPVAIRTLGRLQELGFHNNNIKAIPEKAFMGNPLLQTIHFYDNPIQFVGRSAFQYLPKLHTLSLNGAMDIQEFPDLKGTTSLEILTLTRAGIRLLPSGMCQQLPRLRVLELSHNQIEELPSLHRCQKLEEIGLQHNRIWEIGADTFSQLSSLQALDLSWNAIRSIHPEAFSTLHSLVKLDLTDNQLTTLPLAGLGGLMHLKLKGNLALSQAFSKDSFPKLRILEVPYAYQCCPYGMCASFFKASGQWEAEDLHLDDEESSKRPLGLLARQAENHYDQDLDELQLEMEDSKPHPSVQCSPTPGPFKPCEYLFESWGIRLAVWAIVLLSVLCNGLVLLTVFAGGPVPLPPVKFVVGAIAGANTLTGISCGLLASVDALTFGQFSEYGARWETGLGCRATGFLAVLGSEASVLLLTLAAVQCSVSVSCVRAYGKSPSLGSVRAGVLGCLALAGLAAALPLASVGEYGASPLCLPYAPPEGQPAALGFTVALVMMNSFCFLVVAGAYIKLYCDLPRGDFEAVWDCAMVRHVAWLIFADGLLYCPVAFLSFASMLGLFPVTPEAVKSVLLVVLPLPACLNPLLYLLFNPHFRDDLRRLRPRAGDSGPLAYAAAGELEKSSCDSTQALVAFSDVDLILEASEAGRPPGLETYGFPSVTLISCQQPGAPRLEGSHCVEPEGNHFGNPQPSMDGELLLRAEGSTPAGGGLSGGGGFQPSGLALLHTY SEQ ID NO:11 humanGPR40 nucleic acid sequence atggacctgc ccccgcagct ctccttcggc ctctatgtggccgcctttgc gctgggcttc  61 ccgctcaacg tcctggccat ccgaggcgcg acggcccacgcccggctccg tctcacccct 121 agcctggtct acgccctgaa cctgggctgc tccgacctgctgctgacagt ctctctgccc 181 ctgaaggcgg tggaggcgct agcctccggg gcctggcctctgccggcctc gctgtgcccc 241 gtcttcgcgg tggcccactt cttcccactc tatgccggcgggggcttcct ggccgccctg 301 agtgcaggcc gctacctggg agcagccttc cccttgggctaccaagcctt ccggaggccg 361 tgctattcct ggggggtgtg cgcggccatc tgggccctcgtcctgtgtca cctgggtctg 421 gtctttgggt tggaggctcc aggaggctgg ctggaccacagcaacacctc cctgggcatc 481 aacacaccgg tcaacggctc tccggtctgc ctggaggcctgggacccggc ctctgccggc 541 ccggcccgct tcagcctctc tctcctgctc ttttttctgcccttggccat cacagccttc 601 tgctacgtgg gctgcctccg ggcactggcc cgctccggcctgacgcacag gcggaagctg 661 cgggccgcct gggtggccgg cggggccctc ctcacgctgctgctctgcgt aggaccctac 721 aacgcctcca acgtggccag cttcctgtac cccaatctaggaggctcctg gcggaagctg 781 gggctcatca cgggtgcctg gagtgtggtg cttaatccgctggtgaccgg ttacttggga 841 aggggtcctg gcctgaagac agtgtgtgcg gcaagaacgcaagggggcaa gtcccagaag 901 taa SEQ ID NO:12 human GPR40 amino acidsequenceMDLPPQLSFGLYVAAFALGFPLNVLAIRGATAHARLRLTPSLVYALNLGCSDLLLTVSLPLKAVEALASGAWPLPASLCPVFAVAHFFPLYAGGGFLAALSAGRYLGAAFPLGYQAFRRPCYSWGVCAAIWALVLCHLGLVFGLEAPGGWLDHSNTSLGINTPVNGSPVCLEAWDPASAGPARFSLSLLLFFLPLAITAFCYVGCLRALARSGLTHRRKLRAAWVAGGALLTLLLCVGPYNASNVASFLYPNLGGSWRKLGLITGAWSVVLNPLVTGYLGRGPGLKTVCAARTQGGKSQK SEQ ID NO:13 human GPR43nucleic acid sequence atgctgccgg actggaagag ctccttgatc ctcatggcttacatcatcat cttcctcact ggcctccctg ccaacctcct ggccctgcgg gcctttgtggggcggatccg ccagccccag cctgcacctg tgcacatcct cctgctgagc ctgacgctggccgacctcct cctgctgctg ctgctgccct tcaagatcat cgaggctgcg tcgaacttccgctggtacct gcccaaggtc gtctgcgccc tcacgagttt tggcttctac agcagcatctactgcagcac gtggctcctg gcgggcatca gcatcgagcg ctacctggga gtggctttccccgtgcagta caagctctcc cgccggcctc tgtatggagt gattgcagct ctggtggcctgggttatgtc ctttggtcac tgcaccatcg tgatcatcgt tcaatacttg aacacgactgagcaggtcag aagtggcaat gaaattacct gctacgagaa cttcaccgat aaccagttggacgtggtgct gcccgtgcgg ctggagctgt gcctggtgct cttcttcatc cccatggcagtcaccatctt ctgctactgg cgttttgtgt ggatcatgct ctcccagccc cttgtgggggcccagaggcg gcgccgagcc gtggggctgg ctgtggtgac gctgctcaat ttcctggtgtgcttcggacc ttacaacgtg tcccacctgg tggggtatca ccagagaaaa agcccctggtggcggtcaat agccgtggtg ttcagttcac tcaacgccag tctggacccc ctgctcttctatttctcttc ttcagtggtg cgcagggcat ttgggagagg gctgcaggtg ctgcggaatcagggctcctc cctgttggga cgcagaggca aagacacagc agaggggaca aatgaggacaggggtgtggg tcaaggagaa gggatgccaa gttcggactt cactacagag tag SEQ ID NO:14human GPR43 amino acid sequenceMLPDWKSSLILMAYIIIFLTGLPANLLALRAFVGRIRQPQPAPVHILLLSLTLADLLLLLLLPFKIIEAASNFRWYLPKVVCALTSFGFYSSIYCSTWLLAGISIERYLGVAFPVQYKLSRRPLYGVIAALVAWVMSFGHCTIVIIVQYLNTTEQVRSGNEITCYENFTDNQLDVVLPVRLELCLVLFFIPMAVTIFCYWRFVWIMLSQPLVGAQRRRRAVGLAVVTLLNFLVCFGPYNVSHLVGYHQRKSPWWRSIAVVFSSLNASLDPLLFYFSSSVVRRAFGRGLQVLRNQGSSLLGRRGKDTAEGTNEDRGVGQGEGMPSS DFTTESEQ ID NO:15 human TGR18 nucleic acid sequenceATGATGGCAGAACCATTTACTGAAATTGGTGGATATGCTGCAGGCTTGGCATGGAATGCAACTTGCAAAAACTGGCTGGCAGCAGAGGCTGCCCTGGAAAAGTACTACCTTTCCATTTTTTATGGGATTGAGTTCGTTGTGGGAGTCCTTGGAAATACCATTGTTGTTTACGGCTACATCTTCTCTCTGAAGAACTGGAACAGCAGTAATATTTATCTCTTTAACCTCTCTGTCTCTGACTTAGCTTTTCTGTGCACCCTCCCCATGCTGATAAGGAGTTATGCCAATGGAAACTGGATATATGGAGACGTGCTCTGCATAAGCAACCGATATGTGCTTCATGCCAACCTCTATACCAGCATTCTCTTTCTCACTTTTATCAGCATAGATCGATACTTGATAATTAAGTATCCTTTCCGAGAACACCTTCTGCAAAAGAAAGAGTTTGCTATTTTAATCTCCTTGGCCATTTGGGTTTTAGTAACCTTAGAGTTACTACCCATACTTCCCCTTATAAATCCTGTTATAACTGACAATGGCACCACCTGTAATGATTTTGCAAGTTCTGGAGACCCCAACTACAACCTCATTTACAGCATGTGTCTAACACTGTTGGGGTTCCTTATTCCTCTTTTTGTGATGTGTTTCTTTTATTACAAGATTGCTCTCTTCCTAAAGCAGAGGAATAGGCAGGTTGCTACTGCTCTGCCCCTTGAAAAGCCTCTCAACTTGGTCATCATGGCAGTGGTAATCTTCTCTGTGCTTTTTACACCCTATCACGTCATGCGGAATGTGAGGATCGCTTCACGCCTGGGGAGTTGGAAGCAGTATCAGTGCACTCAGGTCGTCATCAACTCCTTTTACATTGTGACACGGCCTTTGGCCTTTCTGAACAGTGTCATCAACCCTGTCTTCTATTTTCTTTTGGGAGATCACTTCAGGGACATGCTGATGAATCAACTGAGACACAACTTCAAATCCCTTACATCCTTTAGCAGATGGGCTCATGAACTCCTACTTTCATTCAGAGAAAAGTGA SEQ IDNO:16 human TGR18 amino acid sequenceMMAEPFTEIGGYAAGLAWNATCKNWLAAEAALEKYYLSIFYGIEFVVGVLGNTIVVYGYIFSLKNWNSSNIYLFNLSVSDLAFLCTLPMLIRSYANGNWIYGDVLCISNRYVLHANLYTSILFLTFISIDRYLIIKYPFREHLLQKKEFAILISLAIWVLVTLELLPILPLINPVITDNGTTCNDFASSGDPNYNLIYSMCLTLLGFLIPLFVMCFFYYKIALFLKQRNRQVATALPLEKPLNLVIMAVVIFSVLFTPYHVMRNVRIASRLGSWKQYQCTQVVINSFYIVTRPLAFLNSVINPVFYFLLGDHFRDMLMNQLRHNFKSLTSFSRWAHELLLSFREK SEQ ID NO:17 human TGR164 nucleic acid sequenceATGAATGAGCCACTAGACTATTTAGCAAATGCTTCTGATTTCCCCGATTATGCAGCTGCTTTTGGAAATTGCACTGATGAAAACATCCCACTCAAGATGCACTACCTCCCTGTTATTTATGGCATTATCTTCCTCGTGGGATTTCCAGGCAATGCAGTAGTGATATCCACTTACATTTTCAAAATGAGACCTTGGAAGAGCAGCACCATCATTATGCTGAACCTGGCCTGCACAGATCTGCTGTATCTGACCAGCCTCCCCTTCCTGATTCACTACTATGCCAGTGGCGAAAACTGGATCTTTGGAGATTTCATGTGTAAGTTTATCCGCTTCAGCTTCCATTTCAACCTGTATAGCAGCATCCTCTTCCTCACCTGTTTCAGCATCTTCCGCTACTGTGTGATCATTCACCCAATGAGCTGCTTTTCCATTCACAAAACTCGATGTGCAGTTGTAGCCTGTGCTGTGGTGTGGATCATTTCACTGGTAGCTGTCATTCCGATGACCTTCTTGATCACATCAACCAACAGGACCAACAGATCAGCCTGTCTCGACCTCACCAGTTCGGATGAACTCAATACTATTAAGTGGTACAACCTGATTTTGACTGCAACTACTTTCTGCCTCCCCTTGGTGATAGTGACACTTTGCTATACCACGATTATCCACACTCTGACCCATGGACTGCAAACTGACAGCTGCCTTAAGCAGAAAGCACGAAGGCTAACCATTCTGCTACTCCTTGCATTTTACGTATGTTTTTTACCCTTCCATATCTTGAGGGTCATTCGGATCGAATCTCGCCTGCTTTCAATCAGTTGTTCCATTGAGAATCAGATCCATGAAGCTTACATCGTTTCTAGACCATTAGCTGCTCTGAACACCTTTGGTAACCTGTTACTATATGTGGTGGTCAGCGACAACTTTCAGCAGGCTGTCTGCTCAACAGTGAGATGCAAAGTAAGCGGGAACCTTGAGCAAGCAAAGAAAATTAGTTACTCAAACAACCCTTGA SEQ ID NO:18 human TGR164 aminoacid sequenceMNEPLDYLANASDFPDYAAAFGNCTDENIPLKMHYLPVIYGIIFLVGFPGNAVVISTYIFKMRPWKSSTIIMLNLACTDLLYLTSLPFLIHYYASGENWIFGDFMCKFIRFSFHFNLYSSILFLTCFSIFRYCVIIHPMSCFSIHKTRCAVVACAVVWIISLVAVIPMTFLITSTNRTNRSACLDLTSSDELNTIKWYNLILTATTFCLPLVIVTLCYTTIIHTLTHGLQTDSCLKQKARRLTILLLLAFYVCFLPFHILRVIRIESRLLSISCSIENQIHEAYIVSRPLAALNTFGNLLLYVVVSDNFQQAVCSTVRCKVSGNLEQAKKISYSNNP

1. A method of identifying a modulator of a TGR18 polypeptide that hasG-protein coupled receptor activity and (A) comprises at least 70% aminoacid sequence identity to SEQ ID NO:16 (B) consists of at least 50contiguous amino acids of SEQ ID NO:16, or (C) comprises the amino acidsequence of SEQ ID NO:16; wherein the method comprises: contacting thepolypeptide with: a candidate modulator compound and succinic acid; anddetermining the level of activity of the polypeptide in comparison tothe level of activity of the polypeptide in the absence of succinicacid.
 2. The method of claim 1, wherein the step of determining thelevel of activity comprises a competitive assay.
 3. The method of claim1, wherein the compound is contacted with the polypeptide before thesuccinic acid is contacted with the polypeptide.
 4. The method of claim1, wherein the step of determining the level of activity comprises abinding assay.
 5. The method of claim 1, wherein the TGR18 polypeptideis recombinant.
 6. A method of treating a patient with aTGR18-associated disorder, the method comprising administering atherapeutically effective amount of a compound identified using themethod of claim
 1. 7. The method of claim 6, wherein theTGR18-associated disorder is a renal disease.
 8. A method of identifyinga modulator of a TGR164 polypeptide that has G-protein coupled receptoractivity and (A) comprises at least 70% amino acid sequence identity toSEQ ID NO:18 (B) consists of at least 50 contiguous amino acids of SEQID NO:18, or (C) comprises the amino acid sequence of SEQ ID NO:18;wherein the method comprises: contacting the polypeptide with: acandidate modulator compound and α-keto-glutaric acid; and determiningthe level of activity of the polypeptide in comparison to the level ofactivity of the polypeptide in the absence of α-keto-glutaric acid. 9.The method of claim 8, wherein the step of determining the level ofactivity comprises a competitive assay.
 10. The method of claim 8,wherein the compound is contacted with the polypeptide before theα-keto-glutaric acid is contacted with the polypeptide.
 11. The methodof claim 8, wherein the step of determining the level of activitycomprises a binding assay.
 12. The method of claim 8, wherein the TGR164polypeptide is recombinant.
 13. A method of treating a patient with aTGR164-associated disorder, the method comprising administering atherapeutically effective amount of a compound identified using themethod of claim
 8. 14. The method of claim 13, wherein theTGR164-associated disorder is a renal disease.